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1 Karolinska Institutet, Department of Medicine at Center for Molecular Medicine (L8 : 01), Karolinska University Hospital Solna, S-171 76 Stockholm, Sweden
2 The Royal Institute of Technology, Department of Biotechnology, Alba Nova University Centre, Stockholm, Sweden
Correspondence
Mats A. A. Persson
mats.persson{at}ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM113975 and AM113976.
A supplementary table showing sequences of the oligonucleotides used for mutagenesis, in vitro transcriptions and construction of the different vectors is available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Between the two regions there is also a small protein, p7, but the question about to which region it belongs to is still disputed.
Little is known about the details of HCV's replication process, but it is generally believed that most of the NS proteins are involved. This assumption is based on comparisons with other members of the family Flaviviridae and the fact that all NS proteins are localized together with newly synthesized viral RNA on the endoplasmic reticulum (ER) or membranes originating from it (El-Hage & Luo, 2003; Gosert et al., 2003; Mottola et al., 2002). Most NS proteins also interact with several other NS proteins, and some of them interact with all (Dimitrova et al., 2003).
The NS2 protein is an autoprotease that, together with NS3, digests the junction between them (Hijikata et al., 1993). NS2 has been shown to interact with all other NS proteins, but is not required for replication in a subgenomic replicon (Blight et al., 2000; Dimitrova et al., 2003; Lohmann et al., 1999). The NS3 protein carries three different functions: a serine-type protease, an NTPase and a helicase (Gallinari et al., 1998; Grakoui et al., 1993; Kim et al., 1995). NS4A is a cofactor to the NS3 protease, and together they release NS3, NS4A, NS4B, NS5A and NS5B from the polyprotein (Bartenschlager et al., 1995; Grakoui et al., 1993). NS5A has unknown functions, but is indispensable for replication, and NS5B is the RNA-dependent RNA polymerase (Appel et al., 2005; Behrens et al., 1996).
NS4B is essential for HCV replication, but its exact mode of action is unknown (Appel et al., 2005). Even though the amino acid sequence of this protein varies considerably between different genotypes, single amino acid mutations can still affect replication, both positively and negatively (Lindström et al., 2006). When expressed in cells, NS4B induces intracellular membrane changes that are visible by electron microscopy. These structures have been called a ‘membranous web’ (Egger et al., 2002). Rearrangement of the intracellular membranes can also be seen by light microscopy, giving rise to a speckled pattern called membrane-associated foci (MAF), which have been interpreted as a subsequent result of the process that initially gave rise to the membranous web (Egger et al., 2002; Gretton et al., 2005; Kim et al., 1999; Lundin et al., 2003). The MAF have been shown to be the locale for virus replication (Gosert et al., 2003).
Based on in vitro studies, we have previously suggested a topology model for NS4B in the ER membrane. According to this model, NS4B achieves four transmembrane domains immediately after translation with its N and C termini in the cytosol, where processing from NS4A and NS5A is expected to occur. After processing, a rearrangement of NS4B occurs, giving some of the proteins a fifth transmembrane segment, with the N-terminal end in the ER lumen (Lundin et al., 2003). This model has raised concerns about whether the N-terminal rearrangement also occurs in a cellular context, and if the initial topology holds two or four transmembrane domains (Elazar et al., 2004; Moradpour et al., 2003). In the present study, we have focused on the translocation of the N-terminal tail when NS4B is processed from adjacent NS proteins in cells. In addition, we have investigated the role of the recently reported N-terminal amphipathic helix (AH) in the mentioned translocation, and whether the translocation occurs in all major genotypes (Elazar et al., 2004). Finally, we have experimentally settled the issue of whether there are two or four initial transmembrane segments.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To create plasmids for polyprotein processing, all inserts were cloned into the pcDNA3.1(+) vector (Invitrogen). pNS3-4B was made by amplifying the region of interest by using H77c-3396Bam-f and NS4B-3c as primers and pCV/H77C as a template (kindly provided by J. Bukh, NIAID, NIH, Bethesda, MD, USA; Yanagi et al., 1997). pNS3-NST4B was made by assembly PCR (Ho et al., 1989): the NS4B gene was amplified from pCV/H77C by using H77c-5483NST-f and NS4B-3c as primers (PCR1) and the NS3–4A region was amplified from the same template by using primers H77c-3396Bam-f and H77c-5522NST-r (PCR2). The products from both PCRs were purified in agarose gels and used as templates in asymmetrical PCRs with NS4B-3c (for PCR1) and H77c-3396Bam-f (for PCR2) as primers and the Expand Long Template PCR system (Roche) as method of amplification. The products from the two asymmetrical PCRs were assembled by mixing them in equimolar amounts and starting a new amplification with the Expand Long Template PCR system. The resulting product was run in agarose gels and a band of the expected size was excised, purified, digested and ligated into pcDNA3.1(+). To create pNS3-4B/E210, mutations and insertion of the EC4 loop were made as described for pNS4B/E210 (Lundin et al., 2003), but using pNS3-4B as template. pNS3-5B and pNS3-NST5B were made by cutting pCV/H77c with Bsu36I and XbaI to obtain the nucleotide sequences for NS5A, NS5B and the 3' untranslated region from the original vector. The insert was cloned into pNS3-4B and pNS3-NST4B by using the same restriction sites. To create pNS3-NST5A, NS5B was removed from the pNS3-NST5B construct by using the internal cleavage site BspE1 at the end of NS5A, and XbaI. It was replaced with a PCR product encoding the NS5A fragment located downstream of BspE1 and a stop codon. The PCR was performed by using H77C as a template and NS5A-BspE1-f and NS5A-stop-r as primers.
pNS5A was made by amplifying NS5A from pCV/H77C using primers NS5AstartATG/SpeI and NS5Astop/NotI. Inserts were subsequently cut with SpeI and NotI and cloned into pcDNA3.1(+) that had been cut with NheI and NotI.
Plasmids containing NS4B of genotypes 1–6 were created as follows. Genotype 1b: the HCV subgenomic replicon vector pHCVrep1b.BB7 was kindly supplied by C. M. Rice, Rockefeller University, New York City, NY, USA (Blight et al., 2000). Genotype 2b: NS4B was amplified from patient serum by using nested PCR and primers NS4B2b-kpn-f and NS4B2b-not-r for final amplification and cloned into pcDNA3.1(+) (GenBank accession no. AM113975
[GenBank]
). Genotype 3a: NS4B was amplified from patient serum by using a QIAamp viral RNA mini kit (Qiagen). A gene-specific oligonucleotide, NS4B-3a-not-r, was used for cDNA synthesis and together with NS4B3a-kpn-f for the following PCR amplification. The amplified product was cloned into pcDNA3.1(+) (GenBank accession no. AM113976
[GenBank]
). Genotypes 4a, 5a and 6a, cloned into plasmid vectors, were kindly supplied by R. M. Elliott, Institute of Virology, Glasgow University, Glasgow, UK (GenBank accession nos Y11604
[GenBank]
, Y13184
[GenBank]
and Y12083
[GenBank]
; Adams et al., 1997; Chamberlain et al., 1997a, b). The vectors contained NS4B as parts of larger pieces of the HCV genome inserted into pUC119 or pTZ. For genotype 6a, the first 30 nt of NS4B was missing, but by using primers NS4B6a(+30)-kpn-f and NS4B6a-not-r, we amplified NS4B and added the missing nucleotides. The PCR product was cloned into pcDNA3.1(+).
pNST-NS4B-EGFP was made by amplifying the NS4B region from the pNS3-NST4B vector described above by using primers NS4B-5 and NS4B-3b. The product was cloned into pEGFP-N3 (Clontech) by using BamHI and HindIII restriction sites. pNS4B(1b)-EGFP and pAHmut1b-EGFP are described elsewhere (Lindström et al., 2006). In this reference, pAHmut1b-EGFP is called ‘NS4B 1b AHmut-EGFP’.
When making pNS4B/E112long, the EC4 loop (Popov et al., 1997) was first amplified by using primers EC4-STS-f and EC4-STS-r in a PCR with pNS4B/E112 as template (Lundin et al., 2003). The original EC4 was removed from the pNS4B/E112 plasmid by using EcoRI and replaced with the longer EC4 loop by using the same restriction site.
In vitro transcription and translation.
PCR products that included the T7 promoter were used as templates for in vitro transcription. In the genotype study, a glycosylation motif encoding NST (asparagine–serine–threonine) was also added with the 5' primer. In the PCR amplifications, the following templates and primers were used: pNS4B/E112 and pNS4B/E112/EC4long with primers T7-NS4B1a-f2 and NS4B1a-r; pHCVrep1b.BB7 with primers NS4B1b-T7nst-f and NS4B1a-r; pNS4B2b with NS4B2b-T7nst-f and NS4B2b-notI-r; pNS4B3a with NS4B3a-T7nst-f and NS4B3a-notI-r; puc119(4a5227–9355) with NS4B4a-T7nst-f and NS4B4a-r; puc119(5a5366–8334) with NS45a-T7nst-f and NS45a-r; pNS4B6a with NS4B6a-T7nst-f and NS4B6a-not-r. Transcription and translation reactions, as well as their analysis by SDS-PAGE, were done as described previously (Lundin et al., 2003).
Cells, transfection and fluorescence microscopy.
COS-7 and Huh-7 cells [from the ATCC and the Japanese Collection of Research Bioresources (JCRB), respectively] were grown in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum, 100 U penicillin ml–1 and 100 µg streptomycin ml–1 (all reagents were from Invitrogen). Cells were transfected with FuGENE 6 (Roche) according to the manufacturer's protocol. For each plate (60 mm in diameter), we used 2 µg DNA and 10 µl FuGENE 6, except in the trans-complementing study, where we used 1.5 µg polyprotein plasmid and 1 µg pNS5A. For studying NS4B–EGFP, NST–NS4B–EGFP, NS4B1b–EGFP and AHmut1b–EGFP under the microscope, Huh-7 cells were seeded onto coverslips before transfection. At 15, 24 or 48 h post-transfection, the cells were fixed with 4 % formaldehyde at room temperature for 10 min and stained with DAPI (4'6'-diamidino-2-phenylindole) for detection of nuclei. The transfected cells were analysed with a Leica fluorescence microscope and photographed with a Leica DFC300 FX camera using Leica QWin V3 software.
Lysis of cells and peptide: N-glycosidase F (PNGase F) treatment of lysates.
At 20–24 h after transfection, the cells were lysed. Cells that had been transfected for the polyprotein study were lysed with Triton X-114 (2 %). The lysis and separation of detergent and aqueous phases were done as described previously (Lundin et al., 2003). Nuclei were removed from the lysates by centrifugation at 500 g for 5 min. Cells that had been transfected with pNST-NS4B-EGFP or pAHmut1b-EGFP and for the trans-complementing study were lysed with 0.5 % Triton X-100, 150 mM NaCl and 50 mM Tris/HCl (pH 7.5). This lysis buffer also included protease inhibitors: 10 µg PMSF ml–1, 51 µg aprotinin ml–1 and 1 mM sodium orthovanadate. Nuclei and cell debris were removed from the lysates by centrifugation at 500 g for 5 min. Before PNGase F treatment of the cell isolates in the polyprotein study, the detergent phases were precipitated with cold 100 % acetone. The pellets were dissolved in denaturing buffer containing 0.5 % SDS and 1 % 
-mercaptoethanol and incubated at 97 °C for 10 min. Each sample was aliquotted into two portions and 1000 U PNGase F (New England Biolabs) was added to one of them, together with the reaction buffer and NP-40 (giving a final concentration of 50 mM sodium phosphate and 1 % NP-40). The reactions were incubated at 37 °C for 1 h. Cell isolates containing NST–NS4B–EGFP and AHmut1b–EGFP were treated similarly, except for the precipitation step: denaturing buffer was instead added directly to the lysate, giving the same concentration of SDS and -mercaptoethanol.
Western blot.
Cell lysates were reduced and run in 12 % Bis/Tris gels with MOPS buffer using the NOVEX NuPAGE electrophoresis system (Invitrogen). Proteins were electroblotted onto a PVDF membrane (Millipore) that was blocked in 5 % milk in PBS with 0.05 % Tween 20. The membrane was incubated with primary antibody for 2 h at room temperature, washed in PBS with 0.05 % Tween 20 and incubated with a horseradish peroxidase-conjugated antibody of appropriate specificity (GE Healthcare) for 1 h at room temperature. After the membrane had been washed, the signal was detected by using the ECL Western blotting analysis system (GE Healthcare) and Kodak BioMax light film. The membrane was stripped in a buffer containing 2 % SDS, 62.5 mM Tris (pH 6.8) and 7 µl -mercaptoethanol ml–1 for 30 min at 60 °C with occasional agitation. Antibodies used for immunostaining were as follows: 4BLUM(B) : AP was an affinity-purified rabbit anti-NS4B antiserum obtained after immunization with the peptide KIMSGEVPSTEDGSC coupled to keyhole limpet haemocyanin. Rabbit sera 43-6 (anti-NS3), 44-5 (anti-NS4B) and 45-6 (anti-NS5A) were kindly provided by M. Isaguliants, Swedish Institue for Infectious Disease Control, Stockholm, Sweden. Mouse monoclonal anti-NS5B antibody 5B-3B1was a generous gift from D. Moradpour, Centre Hospitalier Universitaire Vaudoism Lausanne, Switzerland (Moradpour et al., 2002). The Western blots were quantified by using Image Gauge v3.45 software (Fuji Science Lab 99/2001). Glycosylation efficiency was calculated as the intensity of the glycosylated band divided by the summed intensities of the glycosylated and non-glycosylated bands.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
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). We wanted therefore to study the translocation in the context of the polyprotein when expressed in cells.
First, three DNA constructs encoding the polyprotein NS3–4A–4B were expressed in COS-7 cells: (i) pNS3–4B encoded the unmodified polyprotein; (ii) pNS3–NST4B included the glycosylation motif, NST, in the N-terminal tail of NS4B; (iii) pNS3–4B/E210 had the EC4 loop [including a glycosylation motif (Popov et al., 1997)] inserted in the C-terminal tail of NS4B. This construct served as negative glycosylation control, ensuring that glycosylation of the N terminus would not be due to general misfolding of the protein. We used Triton X-114 as detergent for the lysis, as this enabled us to separate and concentrate the membrane proteins from the cell extracts (Bordier, 1981). The membrane proteins (detergent phase after separation) were analysed by Western blotting, where glycosylation could be detected as a 2 kDa shift. When the blot was stained for NS4B, it was obvious that NS4B from the pNS3-NST4B construct was well glycosylated, whereas the negative control NS4B/E210 was not [Fig. 2a, top panel, lanes 3 and 6; the EC4 loop in NS4B/E210 adds approximately 3 kDa to NS4B, making its unglycosylated version slightly larger than the glycosylated version of NST–NS4B (having no EC4 loop)]. This indicated that the N terminus of NS4B did indeed translocate across the ER membrane when expressed in the context of NS3 and NS4A.
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, top panel, lanes 3 and 5). This was intriguing, as it may indicate that NS5A or NS5B influences the translocation of the N terminus to the ER lumen.
The same Western blot membrane was also probed for NS3, NS5A and NS5B to ascertain the co-expression of these proteins (Fig. 2a). NS5A and NS5B were only visible in lysates from cells transfected with pNS3-5B and pNS3-NST5B. NS3, NS5A and also NS4B/E210 were partly degraded during the membrane-separation process. The bands of lower molecular mass were not visible if the cells were lysed with a conventional Triton X-100-based method that included protease inhibitors (data not included). NS5A was evident as two distinct bands of the correct size, probably corresponding to the two differently phosphorylated variants p56 and p58. None of the antibodies used in the Western blots recognized any precursor proteins, indicating that processing in COS-7 cells was very efficient or, alternatively, that the precursor may be degraded rapidly.
By treating the lysates from cells transfected with pNS3-NST4B and pNS3-NST5B with PNGase F, the extra bands in these lanes were confirmed to be glycosylated versions of NS4B (Fig. 2b). pNS3-NST5B did not express as well as pNS3-NST4B in these experiments, but was clearly visible after longer exposures (Fig. 2b, lower panel).
The glycosylation efficiency of NS4B, as judged by the bands on the Western blots, varied slightly between experiments. This was probably due both to cell-mediated factors and to the use of different antibodies and antibody aliquots. However, the trend that NS4B from the NS3–NST4B polyprotein was glycosylated more efficiently than NS4B from NS3–NS5B was always consistent.
Translocation of the N terminus of NS4B is affected by NS5A
To examine whether NS5A or NS5B was responsible for the less efficient translocation of the N terminus of NS4B, we made yet another construct, pNS3-NST5A. This vector encoded the polyprotein NS3–4A–4B–NS5A with a glycosylation motif in the N terminus of NS4B. When comparing the expression of this construct with that of pNS3-NST4B and pNS3-NST5B, it was obvious that NS5A was responsible for reducing the translocation of the N terminus of NS4B (Fig. 3a, b). In the presence of NS5A, the translocation efficiency of the N terminus of NS4B was reduced significantly. However, over 15 % of the proteins still relocated their N termini to the ER lumen, which is over 25 % of the total amount of the N terminus translocation seen in the absence of the NS5A. Interestingly, trans-complementation of NS3–NST4B with NS5A did not have the same effect, suggesting that this ability of NS5A only works in cis (Fig. 3c).
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). These mutants were then subjected to in vitro transcription and in vitro translation in the absence and presence of microsomal membranes.
The N-terminal tails of NS4B from all six genotypes translocated into the ER lumen, as reflected by their glycosylation (Fig. 4). Adding a competitive acceptor peptide abolished glycosylation, confirming that we observed true glycosylation.
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NS4B with a disrupted AH in the N terminus does not translocate to the ER lumen
Recently, Elazar and colleagues reported the presence of a putative AH in the N terminus of NS4B that was shown to associate with membranes (Elazar et al., 2004). We therefore wanted to study its role in the translocation process. We introduced the same amino acid changes as Elazar et al. (2004) to disrupt the AH, added a glycosylation motif in the N terminus and a GFP tag to the C terminus (Fig. 5a). The resulting NS4B AH mutant, AHmut1b–EGFP, was expressed in COS-7 cells and the lysates were analysed by Western blot (Fig. 5b). The AH mutant was not glycosylated and, as shown in Figs 1(b) and 4, a glycosylation motif in the N terminus as such does not inhibit translocation. We interpret this as NS4B not being able to translocate its N terminus to the ER lumen when the AH has been disrupted.
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). NS4B1b–EGFP exhibited a speckle-like pattern of MAF that is typical of NS4B and evidence of its membrane-altering ability. AHmut1b–EGFP, however, showed only a reticular pattern typical of the ER, showing that it had lost its ability to rearrange membranes. These data agree with those of Elazar et al. (2004).
The first luminal loop of NS4B is located around aa 112
In our previous work, we determined the topology of NS4B in the ER membrane by using computer predictions and glycosylation mapping, which resulted in a model where NS4B initially has four transmembrane domains (Lundin et al., 2003). A problem with glycosylation mapping is that a minimum distance of 12–14 aa between the acceptor asparagine and the membrane is needed for glycosylation to occur (Nilsson & von Heijne, 1993). As the loops of NS4B were predicted to be very short, an extra 30 aa with a glycosylation motif in the middle (the EC4 loop; Popov et al., 1997) was inserted to ensure enough distance from the membrane. However, we were unable to show consistent glycosylation in the first luminal loop, predicted to be around aa 112 of the protein. We wanted therefore to determine whether the glycosylation loop was slightly misplaced in our mutant pNS4B/E112 (Lundin et al., 2003); this would result in a poorly glycosylated protein, as the distance from the membrane would still be too short. Three extra amino acids, serine–threonine–serine, were added to each end of the EC4 loop (pNS4B/E112long) and we found that NS4B/E112long was glycosylated approximately twofold more efficiently than NS4B/E112 (Fig. 6; quantification data not included). Furthermore, unlike NS4B/E112, the glycosylation of NS4B/E112long was always consistent. This indicated that the first luminal loop of NS4B is located close to, but not exactly at, aa 112 and that the loop is, as predicted, very short.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported that the mutant NS4B/E112, having the EC4 loop in the predicted first luminal loop, was not glycosylated (Lundin et al., 2003). However, the results from this clone were inconsistent and we chose to report them as negative. The topology predictions had indicated that the first luminal loop of NS4B could be expected to be very short. There was therefore an obvious risk that we had inserted the glycosylation loop somewhat askew and not quite at the very top of the loop. This would result in a protein with no or very inefficient glycosylation, as the distance between the membrane and the glycosylation motif would be less than the required 12–14 aa (Nilsson & von Heijne, 1993). By adding 3 aa (S–T–S) at both ends of the EC4 loop at this site, we obtained a protein (NS4B/E112long) that was glycosylated consistently and more efficiently. We interpret this as NS4B indeed having four initial transmembrane segments, and that the first luminal loop of NS4B is located close to, but not exactly at, aa 112. This also concurs with the computer prediction with the constraints that we reported previously (Lundin et al., 2003).
When recombinant NS4B proteins from different genotypes were expressed in vitro, all had the ability to translocate their N termini into the ER lumen (Fig. 4). Elazar and colleagues recently described an AH in the first 29 aa of NS4B that was conserved across all genotypes (Elazar et al., 2004). This AH associated with membranes, which suggested that it may have a role in the translocation of the N terminus across the ER membrane. When we disrupted the AH, the N terminus could no longer relocate to the ER lumen, indicating that a mutated AH seems to inhibit the translocation process. The AH, however, probably does not constitute the fifth transmembrane domain (TMX; Fig. 7), considering the results from our glycosylation studies. First, the NST motif in construct NS4B/E33 is glycosylated, which indicates luminal localization (Lundin et al., 2003). This site is located close to, but downstream of, the AH and should be on the cytosolic side of the ER membrane if the AH was integrated in the membrane. Second, the glycosylation motifs used in this study were located between aa 3 and 5 (in the genotype study) or aa 9 and 11 (in the polyprotein study) of NS4B. If the AH is integrated in the membrane as TMX, these NST sites would be too close to the membrane to be glycosylated. This is also in line with recent results from our group demonstrating that residues located upstream of aa 45 could be glycosylated, whereas those downstream could not (H. Lindström, M. Lundin & M. A. A. Persson, unpublished results). This would indicate that the N terminus of TMX is probably located around aa 60 in the NS4B sequence (Nilsson & von Heijne, 1993).
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). Instead of the normal speckle-like pattern of NS4B, only the reticular staining of a normal ER localization remained (Fig. 5c). This fact, together with our result that the AH mutant is unable to relocate its N terminus to the ER lumen, suggests that the translocation of the N-terminal tail of NS4B is an important step in the process of membrane rearrangements.
We have also studied the translocation of the N terminus of NS4B when expressed and processed from the other NS proteins. In a construct expressing NS3, NS4A and NS4B, the N terminus of NS4B was still glycosylated efficiently when expressed in mammalian cells, indicating luminal localization. However, as the protease NS3, responsible for the cleavage of the NS proteins from the polyprotein, is located on the cytosolic side of the ER membrane, the N terminus of NS4B must be located on that side at the time of processing. Accordingly, the present data support the hypothesis that the N-terminal tail of NS4B must be translocated across the ER membrane by a post-translational mechanism (Lundin et al., 2003).
When NS4B was expressed from polyproteins that also included NS5A, the N terminus was still glycosylated, but with lower efficiency. This indicated that NS5A impairs the translocation of the N terminus to the ER lumen, through either direct or indirect interaction (NS4B has been shown to bind NS5A in several different assays; Dimitrova et al., 2003). Does this imply that the N-terminal tail of NS4B is always retained on the cytosolic side of the ER membrane in an infected cell, as NS5A is always present? We do not think so, as (i) approximately 25 % of the proportion of NS4B that translocated in absence of NS5A (15 vs 60 %) still relocated their N termini in the presence of NS5A (Fig. 3c); (ii) abolishing the translocation of the NS4B N terminus resulted in a protein devoid of membrane-changing capacity (Fig. 5), and (iii) NS4B of all major genotypes had the ability to translocate their N-terminal tails to the ER lumen (Fig. 4). Such a conserved feature generally indicates an important function. A possible scenario could be that NS4B has two different functions during the viral life cycle, where the translocation of the N terminus marks the transition between the two. Evans and colleagues suggested a model in which the hyperphosphorylation of NS5A disrupts the interaction between NS5A and hVAP-A, which stops the replication process (Evans et al., 2004). This would comprise a switch to the next step in the viral life cycle, such as virion assembly and packaging. Maybe the N-terminal tail of NS4B is released at some point from NS5A in a similar mode, and is thereafter free to translocate to the luminal side of the ER membrane, where it can be used for functions required in the later stages of the viral life cycle. Unfortunately, our attempts to study the location of the N terminus in the context of a subgenomic replicon were unsuccessful, as the insertion of a glycosylation motif in the N-terminal tail of NS4B led to a complete loss of replication.
There are several examples of proteins that, upon expression and translocation at the ER, achieve two or more different topologies. Due to this topological heterogeneity, many of these proteins have more than one function (Hegde & Lingappa, 1999; Levy, 1996). One example is the hepatitis B virus L protein. Just as for NS4B, it is integrated co-translationally into the ER membrane, but during maturation, about half of the L molecules translocate their N termini post-translationally into the lumen (Bruss et al., 1994; Lambert & Prange, 2001; Ostapchuk et al., 1994; Prange & Streeck, 1995). It thereby achieves a dual topology where both isoforms have important, but different, functions in the viral life cycle (Bruss & Vieluf, 1995; Le Seyec et al., 1998, 1999; Neurath et al., 1986). It was also reported recently that another HCV protein, p7, can adopt two different topologies in the ER membrane (Isherwood & Patel, 2005).
In conclusion, the present results support a model of the topology of NS4B in which the N-terminal tail translocates to the ER lumen in a post-translational process, evident in all genotypes and also when processed from other NS proteins.
There have been many reports of the effects of different mutations in NS4B, and also different features of the protein that may affect its overall function. In Fig. 7, we have summarized some of these and placed them according to our topology model. The adaptive mutations depicted have all been selected in the subgenomic replicon system and are for higher replication efficiency, a new tropism or interferon resistance (Einav et al., 2004; Elazar et al., 2004; Guo et al., 2001; Koch & Bartenschlager, 1999; Lohmann et al., 2001, 2003; Namba et al., 2004; Zhu et al., 2003). Recent data from our group indicate that single amino acid substitutions in the N terminus of NS4B can have a great impact on replication, both positively and negatively (Lindström et al., 2006). In the present paper, we show that translocation of the N terminus may be influenced by the presence of NS5A and may be associated with the ability of NS4B to rearrange the intracellular membranes. Together, these facts suggest an important role for the N terminus of NS4B in the viral life cycle.
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ACKNOWLEDGEMENTS |
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Received 17 May 2006;
accepted 26 July 2006.
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