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Journal of Virology, June 2005, p. 7911-7917, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7911-7917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Nonstructural Protein Membrane Anchor Deletion Mutants Expressed in the Context of the Hepatitis C Virus Polyprotein

Rainer Gosert,1,2,{dagger} Wiebke Jendrsczok,1,{dagger} Jan Martin Berke,1,3 Volker Brass,1 Hubert E. Blum,1 and Darius Moradpour1,3*

Department of Medicine II, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany,1 Institute for Medical Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel,2 Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland3

Received 20 July 2004/ Accepted 18 February 2005


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ABSTRACT
 
Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.


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TEXT
 
Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral nonstructural proteins, replicating RNA, and altered host cell membranes (6, 11, 20, 31). A specific membrane alteration, designated the membranous web, was recently found to harbor the HCV replication complex (6, 11). Determinants for membrane association of HCV nonstructural proteins have been mapped (reviewed in references 5 and 24). However, the protein-protein interactions involved in formation of a functional replication complex are poorly understood.

HCV nonstructural protein 3 (NS3) is a multifunctional protein with a serine protease domain located in the N-terminal one-third and an NTPase/RNA helicase domain in the C-terminal two-thirds. NS3 by itself has no membrane anchor, but it forms a noncovalent complex with the central domain of the cofactor polypeptide NS4A (18), an interaction that also serves to stabilize NS3 (33, 35). The NS3-4A complex is targeted to membranes by a predicted {alpha}-helix in the N-terminal domain of NS4A (35). NS5A is a phosphoprotein of unknown function that associates with the cytosolic leaflet of the endoplasmic reticulum membrane by a N-terminal amphipathic {alpha}-helix (2, 7, 26). The NS5B RNA-dependent RNA polymerase (RdRp) is a member of the tail-anchored protein family (30). In this case, the C-terminal 21 amino acid (aa) residues, which are dispensable for polymerase activity in vitro (9, 36), traverse the phospholipid bilayer as a transmembrane segment and anchor the RdRp to the cytosolic side of the endoplasmic reticulum membrane (16, 22, 30). Deletion of the membrane anchor domains of NS3-4A, NS5A, or NS5B results in loss of membrane association and diffuse distribution with nuclear accumulation when such truncated proteins are expressed alone (2, 30, 35).

Physical interactions among HCV nonstructural proteins have been described using glutathione S-transferase pulldown assays, coimmunoprecipitation, far Western blot analyses, and conventional yeast two-hybrid screens (4, 10, 27, 32). However, such analyses are notoriously difficult to perform with membrane proteins. Here, we took advantage of the different subcellular localization of membrane-associated and membrane anchor domain deletion-containing HCV nonstructural proteins to investigate whether interactions among the cytosolic domains are sufficiently strong to rescue such truncated proteins to the membrane when expressed in cis in the context of the entire HCV polyprotein.

The subcellular localization of NS3-4A, NS5A, and NS5B proteins with deletions in their membrane anchor domains was investigated first by expression of these proteins alone. An NS3-4A mutant with a deletion of aa 2 to 19 in the N-terminal domain of NS4A (NS3-4A{Delta}N2-19) was prepared by gene splicing by overlap extension (15). Briefly, two DNA fragments were generated by PCR using primer pairs 1-2 and 3-4 and pUHDNS3-4A (35) as template. The two DNA fragments were subsequently joined in a PCR containing primer pair 1-4. The amplification product was digested with NsiI and XbaI, followed by ligation into the NsiI-XbaI sites of pUHDNS3-4A to yield pUHDNS3-4A{Delta}N2-19. This construct allows expression of NS3-4A{Delta}N2-19 from a tetracycline-controlled transactivator (tTA)-dependent promoter (12). An NS5A mutant with a deletion of aa 2 to 21 in the N-terminal amphipathic {alpha}-helix (NS5A{Delta}N2-21) was prepared by PCR using primer pair 6-7 and pUHDNS5Acon as template (2). The amplification product was digested with EcoRI and XbaI, followed by ligation into the EcoRI-XbaI sites of pUHD10-3 (12) to yield pUHDNS5A{Delta}N2-21. The NS5B membrane anchor deletion mutant pCMVNS5Bcon{Delta}C21, lacking the C-terminal 21 aa residues, has been described (30). The U-2 OS human osteosarcoma-derived founder cell line UTA-6 (8), which constitutively expresses the tTA, was transiently transfected with constructs coding for wild-type NS3-4A, NS5A, and NS5B or the corresponding membrane anchor domain deletion mutants, followed by immunofluorescence (IF) analyses using monoclonal antibody (MAb) 1B6 against NS3 (35), 11H against NS5A (2) (kindly provided by Jan Albert Hellings, bioMérieux, Boxtel, The Netherlands), or 5B-12B7 against NS5B (21). Fixation with 4% paraformaldehyde, permeabilization, and immunostaining of cells were performed as previously described (11).

As shown in Fig. 1 (upper panel), NS3-4A, NS5A, and NS5B were found in the cytoplasm in a reticular staining pattern with accumulation in the perinuclear region and staining of the nuclear membrane. In sharp contrast, the membrane anchor deletion mutants lost membrane association and were diffusely distributed throughout the cells (Fig. 1, lower panel). As previously described for similar constructs (2, 30, 35), NS3-4A{Delta}N2-19 and NS5A{Delta}N2-21 were also found in the nucleus with sparing of the nucleoli whereas NS5B{Delta}C21 accumulated in nucleoli, possibly due to the RNA binding properties of the RdRp. Thus, the membrane anchor deletion mutants used in this study showed the expected subcellular distribution when expressed alone.



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  		FIG. 1. Subcellular localization of NS3-4A, NS5A, and NS5B membrane anchor deletion mutants. UTA-6 cells cultured in the absence of tetracycline were transfected with pUHDNS3-4A, pUHDNS5Acon, pCMVNS5Bcon, pUHDNS3-4A{Delta}N2-19, pUHDNS5A{Delta}N2-21, or pCMVNS5Bcon{Delta}C21, as indicated. At 48 h posttransfection, NS3, NS5A, and NS5B were detected by IF analysis using MAbs 1B6, 11H, and 5B-12B7, respectively, and a fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody.

In the next step, deletions of the membrane anchor domains of NS3-4A, NS5A, and NS5B were generated in the context of the HCV polyprotein (Fig. 2). Deletions were carefully designed to preserve functional polyprotein cleavage sites. The NS3-4A mutant was generated by PCR using primer pairs 1-2 and 3-5 (Table 1) and pBRTM/HCV1-3011con (17) (kindly provided by Charles M. Rice, The Rockefeller University, New York, NY) as template. The two DNA fragments were subsequently joined in a PCR containing primer pair 1-5. The amplification product was digested with NsiI and SnaBI, followed by ligation into the same sites of pUHDHCV(H)con (30) to yield pUHDHCV4A{Delta}N2-19. This construct allows expression of the NS3-4A complex with a deletion of aa 2 to 19 in the N-terminal domain of NS4A in the context of the entire polyprotein derived from a functional HCV H strain consensus clone. To delete the membrane anchor of NS5A, two PCR products amplified from pBRTM/HCV1-3011con using primer pairs 8-9 and 10-5 were joined in a PCR containing primer pair 8-5. The amplification product was digested with Bsu36I and SnaBI, followed by ligation into the Bsu36I-SnaBI sites of pUHDHCV(H)con to yield pUHDHCV5A{Delta}N4-21. Finally, the EcoRI-XbaI fragment of pCMVNS5Bcon{Delta}C21 was ligated together with the 7,873-bp EcoRI-EcoRI fragment of pBRTM/HCV1-3011con, encompassing the HCV polyprotein coding region from the start codon to the first one-third of NS5B, into the EcoRI-XbaI sites of pUHD10-3 to yield pUHDHCV5B{Delta}C21.



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  		FIG. 2. HCV nonstructural protein membrane anchor deletion constructs. The HCV polyprotein, encoded by pUHDHCV(H)con, is shown at the top. The membrane anchor domains of NS4A, NS5A, and NS5B are highlighted in gray. The membrane anchor deletion constructs pUHDHCV4A{Delta}N2-19, pUHDHCV5A{Delta}N4-21, and pUHDHCV5B{Delta}C21 are schematically illustrated.


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  		TABLE 1. PCR primers used in this studya

Polyprotein processing of the different full-length constructs was examined by Western blot following transient transfection into UTA-6 cells cultured in the absence of tetracycline. This strategy was chosen because previous experience indicated that the entire HCV polyprotein can be efficiently expressed from a tTA-dependent promoter (25). As shown in Fig. 3, correctly processed core, NS3, NS5A, and NS5B proteins of the expected molecular masses of 21, 70, 56 to 58, and 68 kDa, respectively, could be detected in lysates of cells transfected with pUHDHCV(H)con, indicating robust expression and correct processing of the entire HCV polyprotein in this system. Similarly, transient expression of HCV5A{Delta}N4-21 and HCV5B{Delta}C21 yielded correctly processed proteins of the expected molecular masses. As expected for the HCV4A{Delta}N2-19 mutant, NS3 was detectable only at low levels (Fig. 3, right upper panel), which is consistent with a reduced stability of NS3 expressed without complete NS4A (33, 35). This mutant showed a processing defect with incomplete cleavage of NS5A, presumably at the NS4A-dependent NS4B/NS5A site, and the production of an aberrant smaller-size species. However, the generation of mature NS5A clearly indicates that NS3 was active in the presence of N-terminally truncated NS4A. As anticipated, host cell signal peptidase-mediated processing in the structural region and NS4A-independent cleavage at the NS5A/NS5B site were unaffected, yielding core and NS5B proteins of the expected molecular masses.



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  		FIG. 3. Polyprotein processing of membrane anchor deletion constructs. UTA-6 cells cultured in the absence of tetracycline were transfected with pUHDHCV(H)con, pUHDHCV4A{Delta}N2-19, pUHDHCV5A{Delta}N4-21, or pUHDHCV5B{Delta}C21, as indicated. At 48 h posttransfection, cell lysates were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblot using MAbs C7-50 against core (23) (upper left panel), 1B6 against NS3 (upper right panel), 11H against NS5A (lower left panel), and 5B-3B1 (21) (lower right panel). Nontransfected cells served as controls. Molecular mass markers are shown on the left of each panel.

IF analysis was performed next to investigate whether interactions among the cytosolic domains could rescue the membrane association of nonstructural proteins with deletions of their membrane anchor domains. As shown in Fig. 4 (left panel), core, NS3, NS5A, and NS5B expressed from the wild-type HCV polyprotein were found in the cytoplasm in a reticular staining pattern with accumulation in dot-like structures which, as shown previously (6, 11), represent membranous webs. It is important to note that HCV protein expression is sufficient to induce the formation of membranous webs which in the context of active HCV RNA replication harbor viral replication complexes (6, 11). Consistent with earlier observations (6), a minor fraction of core was also found in the nucleus with accumulation in nucleoli. The distribution of core, NS5A, and NS5B was similar in the case of HCV4A{Delta}N2-19. Possibly due to the processing defect observed by immunoblotting (Fig. 3), the number and size of dot-like structures revealed by the NS5A MAb were reduced compared to the wild-type construct. More importantly, the membrane anchor deletion-containing NS3-4A complex was found in a diffuse staining pattern with even distribution between the cytoplasm and nucleus and sparing of the nucleoli (Fig. 4). This staining pattern was indistinguishable from that of NS3-4A{Delta}N2-19 expressed alone (compare with Fig. 1, lower left panel). Similarly, the membrane anchor deletion-containing NS5A and NS5B proteins expressed by HCV5A{Delta}N4-21 and HCV5B{Delta}C21, respectively, were diffusely distributed and showed the same staining pattern as the corresponding mutants expressed alone (compare Fig. 4 with Fig. 1, lower middle and lower right panels). Membrane association of core, NS3, and wild-type NS5A or NS5B, respectively, as well as the formation of dot-like structures, representing membranous webs, was not significantly affected in these constructs. In conclusion, these results demonstrate that membrane association of the membrane anchor deletion mutants is not rescued by their expression in the context of the HCV polyprotein.



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  		FIG. 4. Subcellular localization of NS3-4A, NS5A, and NS5B membrane anchor deletion mutants expressed in the context of the HCV polyprotein. UTA-6 cells cultured in the absence of tetracycline were transfected with pUHDHCV(H)con, pUHDHCV4A{Delta}N2-19, pUHDHCV5A{Delta}N4-21, or pUHDHCV5B{Delta}C21, as indicated. At 48 h posttransfection, core (C), NS3, NS5A, and NS5B or the respective membrane anchor deletion mutants were examined by IF analysis using MAbs C7-50, 1B6, 11H, and 5B-12B7, respectively.

Membrane flotation analyses were performed to confirm these observations. To this end, cells transfected with the different expression constructs were subjected to hypotonic lysis, followed by equilibrium centrifugation of cell lysates through Nycodenz gradients, as described previously (22, 26). Under these conditions, membrane proteins float to the upper, low-density fractions, while soluble proteins remain in the lower, high-density fractions. As expected, NS3-4A, NS5A, and NS5B proteins expressed from the wild-type polyprotein construct floated to the membrane-containing low-density fractions (Fig. 5, left panels). By contrast, the membrane anchor deletion mutants remained in the high-density fractions, regardless of whether they were expressed alone (middle panels) or in the context of the HCV polyprotein (right panels). Of note, a minor proportion of the NS5A mutant was found in the membrane fractions under both conditions. This is consistent with the idea that one or more additional regions outside of the N-terminal amphipathic {alpha}-helix of NS5A may contribute to its membrane association (26).



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  		FIG. 5. Flotation analyses of membrane anchor deletion mutants. Lysates of UHCVcon-57.3 cells, which inducibly express the wild-type HCV(H)con polyprotein (30), or UTA-6 cells transfected with pUHDNS3-4A{Delta}N2-19, pUHDNS5A{Delta}N2-21, pCMVNS5Bcon{Delta}C21, pUHDHCV4A{Delta}N2-19, pUHDHCV5A{Delta}N4-21, or pUHDHCV5B{Delta}C21 were subjected to equilibrium centrifugation through Nycodenz gradients. Fractions were collected from the top and analyzed by immunoblotting using MAbs 1B6, 8N, 11H, and 5B-3B1 against NS3, NS4A, NS5A, and NS5B, respectively.

Taken together, the IF and membrane flotation analyses shown above demonstrate that interactions of the cytosolic domains of NS3-4A, NS5A, and NS5B with the remaining HCV proteins are not sufficiently strong to rescue membrane anchor deletion mutants to the membrane. These results are in good agreement and further extend similar observations made with a membrane association-deficient NS5A mutant expressed in the context of the HCV nonstructural proteins (7). Thus, membrane association of each nonstructural protein is essential for its incorporation into the replication complex. However, formation of this complex, at least at the resolution of light microscopy, does not depend on the presence of all membrane anchor domains. By contrast, and in agreement with a central role of NS4B in formation of the membranous web (6), we have recently found that deletion of NS4B in the context of the HCV polyprotein abrogates formation of dot-like structures (Valérie Castet and D. Moradpour, unpublished data).

Recent evidence suggests that the HCV RdRp, similar to poliovirus 3D polymerase (13, 19), may form multimers and thereby gain cooperative activity (28, 34). In addition, homotypic interactions have been reported for NS5A (4). This prompted us to investigate whether protein-protein interactions between membrane anchor deletion-containing NS5A and NS5B with their corresponding wild-type counterparts may rescue membrane association of the truncated proteins. To this end, membrane anchor deletion mutants with an engineered hemagglutinin (HA) epitope tag were coexpressed in trans with the HCV polyprotein and their subcellular localization analyzed by IF. In brief, an NS5A fragment with a deletion of aa 2 to 21 in the N-terminal domain and a C-terminal HA tag was amplified from pBRTM/HCV1-3011con by PCR using primer pair 11-12 (Table 1). The amplification product was digested with EcoRI and XbaI, followed by ligation into the EcoRI-XbaI sites of pUHD10-3 to yield pUHDNS5A{Delta}N2-21HA. A C-terminal HA tag was fused to NS5B{Delta}C21 by PCR using primer pair 13-14 and pBRTM/HCV1-3011con as a template, followed by digestion with EcoRI and XbaI and ligation into the EcoRI and XbaI sites of pUHD10-3 to yield pUHDNS5B{Delta}C21HA. To exclude the possibility that the position of the HA tag influences the subcellular localization of NS5B{Delta}C21, a similar construct, pUHDHANS5B{Delta}C21, with an N-terminal HA tag was prepared using primer pair 15-16.

The tagged mutants were transfected into UHCVcon-57.3 cells, which inducibly express the entire HCV polyprotein upon tetracycline withdrawal (30). Double-label IF analysis was subsequently performed to determine the subcellular distribution of the tagged mutants and of NS3 as a marker for a membrane-associated protein. As shown in Fig. 6, NS3 expressed from the inducible transgene appeared in the typical reticular staining pattern and in cytoplasmic dot-like structures irrespective of the coexpressed mutant (upper panel). In contrast, HA-tagged NS5A{Delta}N2-21 was found in a diffuse staining pattern distributed throughout the cell. Similarly, NS5B{Delta}C21 was diffusely distributed and accumulated in the nucleus, irrespective of the position of the HA tag. Interestingly, the HA tag seemed to prevent nucleolar accumulation of this mutant. Taken together, these results indicate that homotypic interactions of NS5A or NS5B coexpressed in trans are relatively weak. Identical results were obtained when the tagged mutants were cotransfected with their wild-type counterparts expressed alone or in the context of the HCV polyprotein (data not shown).



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  		FIG. 6. Subcellular distribution of HA-tagged membrane anchor deletion mutants in the presence of the entire wild-type polyprotein. UHCVcon-57.3 cells were derepressed by tetracycline withdrawal and at the same time transiently transfected with pUHDNS5A{Delta}N2-21HA, pUHDHANS5B{Delta}C21, or pUHDNS5B{Delta}C21HA, as indicated. At 48 h posttransfection, the membrane anchor deletion mutants were visualized by a polyclonal anti-HA antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and a Cy3-conjugated goat anti-rabbit secondary antibody. NS3 was detected with MAb 1B6 and an Alexa 488-conjugated goat anti-mouse secondary antibody.

Taken together, our data suggest that interactions among cytosolic domains of NS3-4A, NS5A, and NS5B are relatively weak compared to their membrane association. Membrane association of the individual nonstructural proteins is essential for incorporation into the HCV replication complex. The absence of strong interactions between the viral nonstructural proteins may provide them with a certain flexibility to fulfill their multiple functions during viral replication, e.g., synthesis of positive- and negative-strand RNAs by the RdRp or serine protease and NTPase/RNA helicase activity of the NS3-4A complex (37). These observations, together with the identification of absolutely conserved residues in the membrane anchor domains of NS5A and NS5B that are essential for viral RNA replication, but not for membrane association per se (22, 26), suggest that the membrane anchor domains are central organizing elements in the replication complex and that important protein-protein interactions between these domains may occur within the membrane.

Two points need to be considered, however. First, it is possible that replicating viral RNA mediates interactions that we could not detect in our assays. However, interactions that have thus far been reported have also been found in the absence of a genuine viral RNA. Second, we cannot rule out conformational alterations of nonstructural proteins lacking their membrane anchor domains or, in the case of the experiments shown in Fig. 6, fused to an epitope tag. However, biochemical and structural studies have shown correct folding and functionality of NS3-4A with only the central portion of NS4A present (1, 18, 37). Accordingly, protease activity of NS3-4A{Delta}N2-19 was partially impaired only at the NS4B/NS5A cleavage site. In addition, it was shown that the C-terminal 21 aa of NS5B are dispensable for polymerase activity in vitro (9, 36). No functional or structural information is available for NS5A. Thus, at least the NS3-4A and NS5B membrane anchor deletion mutants should have folded properly.

It is interesting that interactions among cytosolic domains of nonstructural proteins from other positive-strand RNA viruses are essential for incorporation of these proteins into membrane-associated replication complexes; e.g., membrane association of poliovirus 3D polymerase is mediated by protein precursor 3AB (14). Similar to poliovirus 3D, the RdRp of mouse hepatitis virus is directed to the membrane by an as yet unknown viral or virus-induced factor (3). Furthermore, membrane association of the nonstructural polyprotein of Semliki Forest virus is mediated by nsP1 (29). In these instances, membrane association of essential replicase components is ensured by interactions among cytosolic domains of nonstructural proteins. The observation that replication complex incorporation of each nonstructural protein depends on its own membrane anchor domain appears characteristic for HCV. Studies on the determinants and mechanisms of membrane association of nonstructural proteins from pestivirus, GB virus, and flavivirus are in progress to explore whether this feature is shared among different members of the Flaviviridae family.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge Elke Bieck and Anja Wahl for excellent technical assistance; Zsuzsanna Pal for support with the membrane flotation assays; Denise Egger, Kurt Bienz, and François Penin for discussions and critical reading of the manuscript; Charles M. Rice for pBRTM/HCV1-3011con; and Jan Albert Hellings for MAbs 8N and 11H.

This work was supported by grants Mo 799/1-3 from the Deutsche Forschungsgemeinschaft, QLK2-CT-2002-01329 and LSHM-CT-2004-503359 (VIRGIL European Network of Excellence on Antiviral Drug Resistance) from the European Commission, 01 KI 9951 from the Bundesministerium für Bildung und Forschung, and 04C59 from the Novartis Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, CH-1011 Lausanne, Switzerland. Phone: 41 21 314 47 23. Fax: 41 21 314 47 18. E-mail: Darius.Moradpour{at}hospvd.ch. Back

{dagger} R.G. and W.J. contributed equally to this work. Back


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Journal of Virology, June 2005, p. 7911-7917, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7911-7917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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