key: cord-0738839-h9bx7yvt
authors: Romero-Brey, Inés; Berger, Carola; Kallis, Stephanie; Kolovou, Androniki; Paul, David; Lohmann, Volker; Bartenschlager, Ralf
title: NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication
date: 2015-07-07
journal: mBio
DOI: 10.1128/mbio.00759-15
sha: 17bfdf17f46ae63e52c3e7c085bac97057e9ae61
doc_id: 738839
cord_uid: h9bx7yvt

Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.

velope protein 1 (E1) and E2 that build up the virus particle. The viroporin p7 and nonstructural protein 2 (NS2) are accessory factors required for the assembly of infectious HCV particles (reviewed in reference 10). The minimal HCV replicase comprises the remaining nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B (11) . NS3 is composed of two domains comprising an N-terminal serine protease that is activated by interaction with NS4A and responsible for proteolytic maturation of the replicase proteins and an NTPase/RNA helicase domain formed by the C-terminal two-thirds of NS3 (reviewed in reference 12). Highly hydrophobic NS4B is believed to build the scaffold of the viral replication complex and forms oligomeric complexes that are important for the formation of DMVs (13) (14) (15) (16) . NS5A is an RNAbinding phosphoprotein containing an N-terminal amphipathic ␣-helix (AH) that stably tethers the protein to intracellular membranes (17, 18) (reviewed in reference 19) . NS5A is composed of three domains (D1 to D3) that are separated by low-complexity sequence I (LCSI) and LCSII. D1 is the main determinant of RNA replication, whereas D3 plays a major role in the assembly of infectious virus particles, probably by interacting with the core protein (20) (21) (22) (23) . In fact, major parts of D3 can be deleted without affecting RNA replication and D3 tolerates in-frame insertion of heterologous proteins such as green fluorescent protein (GFP) (24) . The exact role of D2 remains to be determined, since a large segment within this domain can be deleted with no significant effect on RNA replication and virus production in cell culture (20) ; however, a recent study has demonstrated that D2 is required to suppress the activation of the interferon response (25) . NS5B is a tail-anchored RNA-dependent RNA polymerase (8) .

HCV RNA replication is linked to membrane alterations that are induced by the concerted action of viral and cellular proteins (reviewed in reference 26) . We have shown earlier that induction of the membranous web requires viral replicase polyprotein NS3-5B. The only protein able to induce DMVs and predominantly multimembrane vesicles on its own was NS5A, but with only very low efficiency compared to that obtained by the expression of an NS3-5B polyprotein (4) . In the present study, we identified the determinants within NS5A required for DMV formation and analyzed the role of polyprotein cleavage in cis and trans for the establishment of the membranous HCV replication factory.

Given the prominent role of NS5A in HCV-induced membrane rearrangements (4), we characterized the involvement of the three NS5A domains in the formation of DMVs and correlated this property with viral RNA replication. To this end, we generated several deletion mutations by removing each NS5A domain individually in the context of genotype 2a isolate JFH-1 (27) (Fig. 1A) . The mutations were introduced into a subgenomic luciferase reporter replicon, and in vitro transcripts were transfected into Huh7 cells. Consistent with our earlier report (20) , we found that only the deletion of a segment of domain 2 (⌬D2short) or all of domain 3 (⌬D3) was tolerated (Fig. 1B) , whereas removal of D1 or all of D2 blocked RNA replication (Fig. 1B) .

To study the impact of these deletion mutations on DMV formation, we used a T7 RNA polymerase-based expression system that is sufficient to induce membranous web formation in the absence of HCV RNA replication (4). Mutations were introduced into an NS3-5B expression construct allowing cytoplasmic ex-pression of the HCV polyprotein (Fig. 1C ). Expression and cleavage of the polyprotein in transfected Huh7-Lunet/T7 cells that stably express the T7 RNA polymerase were determined by Western blotting with different NS5A-monospecific antibodies to account for the removal of epitopes residing in different NS5A domains. As shown in Fig. 1C , the polyproteins were properly processed, with the exception of ⌬D1, where we observed two proteins that, on the basis of their apparent molecular weights and immunoreactivities with the monospecific antisera, were identified as NS5A and an uncleaved NS4B-5A precursor. Nevertheless, we note that fully processed NS4B was also generated by this mutant and the abundance of NS5A and NS4B was comparable to that in the other mutants.

We next determined the capacity of these mutants to induce DMV formation by using electron microscopy (EM) of cryofixed cells that had been transfected with the corresponding expression constructs (Fig. 2) . Two parameters were used for EM analysis. First, the abundance of DMVs per cell profile, which was determined by counting the DMVs residing in the cytoplasmic area (measured in square micrometers and calculated as the cell surface of a given cell section minus the nuclear region, which is devoid of DMVs). To account for the possibility that DMVs reside in different planes, sections taken at different cell levels were included in our quantification. Second, the DMV diameter was measured as the distance between the two farthest opposed points on the membrane of a given DMV to account for the fact that DMVs most often are elliptical. We note that an elliptical rather than a circular appearance of DMVs might be caused by compression artifacts generated during sample fixation or sectioning. However, this possible artifact did not affect our analysis because we always used the wild type as a reference for comparison.

By using this approach, we found that removal of D1 (⌬D1) led to complete abrogation of DMV formation but induced fragmentation and swelling of the ER ( Fig. 2B ; Table 1 ). Interestingly, the deletion mutant that lacked all of D2 (⌬D2long) and was unable to replicate (20) (Fig. 1B) still induced the formation of DMVs ( Fig. 2C ) but with significantly reduced efficiency, as revealed by quantification of the number of DMVs per cell surface area ( Fig. 2I ; Table 1 ). Moreover, in contrast to wild-type NS3-5B, these DMVs exhibited an average diameter of 133 Ϯ 20 nm (n ϭ 90), which is~30 nm smaller than the majority of the DMVs detected in cells expressing wild-type NS3-5B ( Fig. 2H ; Table 1 ). In addition, these DMVs were confined in smaller clusters, in contrast to the more interspersed distribution of wild-typeinduced DMVs.

In the NS3-5B polyprotein containing a small deletion of D2 (⌬D2short), RNA replication was only moderately affected ( Fig. 1B) and DMVs with wild-type morphology were induced ( Fig. 2D and H; Table 1 ). Also, in this mutant, efficiency of DMV formation was reduced ( Fig. 2I ; Table 1 ). Removal of the complete domain 3 (⌬D3) had no effect on RNA replication but selectively abolished the assembly of infectious HCV particles (20) . As expected, this mutation induced DMVs (Fig. 2E ) but with a diameter of 220 Ϯ 52 nm (n ϭ 90), which is~50 nm larger than that of wild-type DMVs ( Fig. 2H ; Table 1 ).

The results described so far suggested that neither D2 nor D3 are required for DMV formation. To corroborate this observation, we generated two additional deletion mutants: ⌬D2D3, lacking both domains and LCSII, and mutant ⌬LCSI-D3, lacking in addition LCSI (Fig. 1A) . Both mutants still induced the formation structures. An enlarged view of the organization of the NS5A domain is shown below. The numbers at the top refer to amino acid residues of the JFH-1 polyprotein, and numbers in parentheses refer to residues of NS5A. Deletion mutants are shown below; the names of the mutants are shown to the left of the illustrations. The numbers in parentheses refer to the amino acid (aa) residues of the polyprotein that were removed. (B) Replication kinetics of NS5A deletion mutants. Huh7.5 cells were transfected with subgenomic Luc reporter replicons. Cells were harvested 4, 24, 48, and 72 h post electroporation (hpe), and the luciferase activity in the lysates was determined. Values were normalized to the 4-h value, reflecting transfection efficiency. A replication-deficient mutant encoding an inactive NS5B polymerase served as a negative control (⌬GDD). RLU, relative light units; WT, wild type; LOD, limit of detection. (C) Polyprotein processing of NS5A mutants. The basic construct used for expression of the NS3-5B polyprotein and NS5A mutant proteins derived therefrom is shown on the top. It contains the promoter (Pm) of the T7 RNA polymerase (gray box) and the IRES of encephalomyocarditis virus upstream of the polyprotein coding region. Huh7-Lunet/T7 cells were transfected with expression constructs specified above the lanes, and 24-h later, cells were lysed and HCV proteins were detected by Western blotting with various NS5A-, NS4B-, and NS3-specific antibodies as specified on the right of each panel. ␤-Actin served as a loading control. The values on the left are the molecular masses of the standards used. The uncleaved NS4B-⌬D15A precursor detected in the NS5A (9E10)-and NS4B-specific immunoblot analysis is indicated by arrowheads. The two phosphorylated variants of wild-type NS5A in the upper panel are labeled with asterisks, and they are referred to as p56 and p58.

of DMVs ( Fig. 2F and G; Table 1 ), arguing that NS5A D1 is the critical determinant of membranous web formation. Consistent with this assumption, when correlative-light EM (CLEM)-based analysis of cells with proven expression of an NS3-5B polyprotein lacking NS5A D1 was used, no DMVs were detected (Fig. 3) . Note that the field of view selected contains many mitochondria and especially lipid droplets (LDs) that were often found randomly clustered in certain regions of a cell. In cells containing large amounts of DMVs, mitochondria were frequently found at the periphery of DMV-containing areas, whereas LDs were found either within these areas or surrounding them (not shown). Taken together, these results showed that the intrinsically disordered regions of NS5A (LCSI up to D3) are dispensable for DMV induction yet required for RNA replication (D2) or virus assembly (D3) (20) , respectively. Thus, the defect of mutant ⌬D2long in RNA replication is not linked to a lack of DMV formation. Conserved residues in the amino-terminal AH and the zincbinding domain of NS5A are essential for the formation of DMVs. To assess further the importance of NS5A D1 and the N-terminal AH for the induction of DMVs, we analyzed a set of previously described mutations within these regions that specifically disturb features of the AH, abrogate zinc binding of subdomain 1a, or disturb the disulfide bond of subdomain 1b (Fig. 4A ). In the AH, which is required for HCV RNA replication (17) , two mutations reported earlier (28) in the context of HCV isolate Con1 (genotype 1) were inserted into the JFH-1 isolate (genotype 2a). In these mutants, originally designated GBV-C 8-13 and GBV-B 8-13, the amphipathic character of the helix was maintained but highly conserved N-terminal amino acid residues 8 to 13 of NS5A were replaced with the homologous sequences of closely related GB virus C (GBV-C) and GBV-B, respectively (Fig. 4A ). In agreement with previous results (28) , the replication of mutant GBV-C 8-13 was clearly lower than that of the wild type (Fig. 4B) , which we found to correlate with reduced efficiency of DMV formation in the expression system ( Fig. 4G ; Table 1 ). Mutant GBV-B 8-13 was replication incompetent (Fig. 4B ) and unable to induce DMVs upon expression of the corresponding NS3-5B polyprotein in Huh7-Lunet/T7 cells, whereas singlemembrane vesicles (SMVs) were still detectable ( Fig. 4D ; Table 1 ). Importantly, steady-state levels of these variant NS5A proteins were comparable to those of the wild type (Fig. 4C) . Thus, specific residues within the NS5A N-terminal AH are required for the biogenesis of DMVs.

NS5A contains a zinc-binding motif in the N-terminal part of The data presented for the wild type are the same as those shown in Fig. 2H and I because the mutant sets shown in Fig. 2 and 4 were analyzed in parallel, along with the wild type. n.d., not determined; n.s., nonsignificant; **, P Ͻ 0.001; ***, P Ͻ 0.0001. D1 (designated subdomain D1a) that is absolutely required for NS5A structure integrity (29) . A C59G mutation affecting the cysteine residue coordinating Zn 2ϩ binding inhibited HCV RNA replication (18) (Fig. 4B) and abrogated the formation of DMVs, whereas SMVs were still detectable (Fig. 4E) . Interestingly, this mutant also displayed a cleavage defect at the NS4B-5A site, but its steady-state level of processed NS5A was comparable to that of the other mutants (Fig. 4C) . In contrast, an alanine substitution for cysteine 142 (C142A), reported to form a disulfide bond within subdomain 1b of NS5A (29), affected neither polyprotein cleavage (Fig. 4C ) nor HCV RNA replication (Fig. 4B) . However, this mutant induced DMVs with lower efficiency than the wild type in the pTM system ( Fig. 4F and G) but not in cells transfected with the replicon (Table 1 ).

In conclusion, these results suggest that both the N-terminal membrane anchor and structural integrity of NS5A D1 are essential prerequisites for DMV formation.

DMV formation requires the NS3 helicase domain. Although we and others have shown earlier that efficient polyprotein cleavage in trans can be achieved by using only the N-terminal NS3 protease domain and the NS4A cofactor (30, 31), we did not know whether this minimal protease was sufficient to induce DMVs or whether full-length NS3 would be required. Therefore, we generated an NS3-5B polyprotein expression construct lacking the helicase domain (Fig. 5A) . Upon the transfection of Huh7-Lunet/T7 cells with this construct, we observed efficient polyprotein cleavage (Fig. 5B) . However, this mutant was unable to induce DMVs and only SMVs were observed (Fig. 5C) . These results show that the helicase domain of NS3 is required for the induction of DMVs. Therefore, all subsequent trans cleavage assays were conducted with full-length NS3.

Expression of NS3-4A in cis is required for efficient formation of DMVs. In order to determine the cis and trans requirements for DMV formation, we established a polyprotein trans cleavage assay. This assay is based on cell lines stably expressing NS3-4A and transient coexpression of an NS4B-5B polyprotein substrate from a separate RNA (Fig. 6A) . As a negative control, we used a cell line stably expressing an inactive full-length NS3-4A protease mutant in which the active-site serine residue had been replaced with an alanine residue (S139A) (32) . Western blot analysis revealed proper polyprotein cleavage by wild-type NS3-4A, whereas only uncleaved precursor and presumably degradation products were found in cells containing the proteolytically inactive NS3-4A protein (Fig. 6B) . While only large SMVs were found in the latter case (Fig. 6D) , vesicles composed of two or more lipid bilayers were detected in cells containing the active NS3-4A protein (Fig. 6C) . However, the number of DMVs induced in this trans cleavage setting was very low compared to that induced by a cis-cleaving NS3-5B polyprotein (0.0063/m 2 versus 1.07/m 2 ; Fig. 6F ). Moreover, vesicles induced upon trans cleavage of the polyprotein had an electron-dense interior, indicative of a protein-rich content, which was not found in DMVs induced by the NS3-5B polyprotein. These results suggested that polyprotein cleavage in cis is required for efficient formation of DMVs and that DMVs detected in the trans cleavage system might represent different structures.

Regulated cleavage at the NS4B-5A junction determines DMV biogenesis. For several RNA viruses, it has been shown that processing intermediates serve distinct functions in the replication cycle. In HCV, polyprotein processing by NS3-4A is a tightly regulated process that occurs in a preferential order with rather protracted cleavage at the NS4B-5A site (33) (34) (35) (36) (37) . Interestingly, mutations altering cleavage kinetics at this site are detrimental for HCV RNA replication, but the underlying mechanism has not been studied (38) . Assuming that cleavage kinetics might play an important role in the biogenesis of the membranous HCV replication factory, we determined the impact of altered NS4B-5A cleavage kinetics on the formation of DMVs. On the basis of a recent report (38) , we generated mutant P43VV, exhibiting accelerated cleavage kinetics due to two valine substitutions for proline and isoleucine residues at the P4 and P3 positions of the cleavage site (Fig. 7A, Fast mutant) . Indeed, when it was expressed in the context of an NS3-5B polyprotein, much less uncleaved NS4B-5A was found with this mutant, consistent with accelerated cleavage at this site (Fig. 7A) . Moreover, consistent with the earlier report (38) , we found that the P43VV double mutation, inserted into a subgenomic replicon, reduced RNA replication up to 100-fold at early time points posttransfection (Fig. 7B ). When it was tested in the context of the NS3-5B polyprotein expression construct, the formation of DMVs (Fig. 7C) with a regular morphology was observed (Fig. 7D) . However, DMV abundance was significantly lower than that in the wild type (Fig. 7E) , arguing that regulated cleavage at the NS4B-5A site is important for efficiency of HCVinduced membrane rearrangements and thus RNA replication.

To corroborate this hypothesis, we expressed an NS3-5B polyprotein lacking any NS4B-5A precursor because of the insertion of a heterologous encephalomyocarditis virus IRES between the NS4B and NS5A coding regions (Fig. 8A ). This construct did not support RNA replication (Fig. 8A) , suggesting that an unprocessed NS4B-5A precursor might play an important role in this process. Expression of the corresponding polyprotein in Huh7-Lunet/T7 cells revealed NS4B abundance to a level comparable to that of the wild type, whereas the abundance of NS5A was slightly reduced but it was still well detectable (Fig. 8B) . Importantly, the IRES insertion mutant did not support DMV formation but instead induced SMVs (Fig. 8C) . These results suggest that regulated cleavage at the NS4B-5A junction plays a pivotal role in the biogenesis of HCV replication factories and that an NS4B-5A precursor appears to be required for DMV formation. 

Positive-strand RNA viruses, including HCV, replicate their genomes in association with intracellular membranes (1). However, it remains largely unknown how viral proteins can promote the formation of these remarkable membrane alterations. In the present study, we explored the role of viral NS proteins in the formation of HCV-induced DMVs. We reported earlier that induction of the membranous web requires the concerted action of all of the HCV replicase proteins (4) . Although the sole expression of NS5A was sufficient to induce vesicles containing two or predominantly more lipid bilayers, DMVs were just sporadically observed. Only when we expressed the polyprotein comprising NS3 to NS5B was DMV formation abundant, arguing for a complex and coordinated interplay between the viral replicase factors to induce these membrane rearrangements. This assumption is supported by the observation that mutations residing, e.g., in NS4B can also drastically reduce DMV formation or alter their morphology even though NS5A is present (16) . Thus, while NS5A can induce DMVs on its own, additional determinants are required for the proper formation of the membranous HCV replication factory. To gain further insights into these determinants, in the present study, we mapped the elements within NS5A that are required for DMV biogenesis. We found that the N-terminal AH and D1 are the primary determinants of this process. This is consistent with the observation that D1 is essential for RNA replication. Moreover, this domain binds RNA (29, 39) , but it remains to be determined whether this property plays a role in DMV formation. The observation that the ⌬D2short mutant does not produce hyperphosphorylated NS5A in spite of replication and DMV formation efficiency comparable to those of the wild type suggests that hyperphosphorylation of NS5A is not strictly required for DMV formation. Although we cannot rule out the possibility that, in the case of this NS5A variant, the basal and hyperphosphorylated forms have very similar electrophoretic mobility and thus are not separated by conventional gel electrophoresis, our conclusion is in keeping with the notion that hyperphosphorylation is critical for assembly, whereas basal phosphorylation appears to favor RNA replication (40) (41) (42) .

We reported earlier that isolated DMVs contain HCV replicase proteins and enzymatically active replicase complexes, arguing that DMVs are the site of HCV replication (7) . Consistent with this assumption, in this study, we observed a correlation between the amount of DMVs detected in cells containing replicating HCV RNA and replication fitness as determined with subgenomic replicons (Table 1) . However, this correlation appeared less strict when ectopic expression of the polyprotein was used to induce DMVs. For instance, the ⌬D2short and C142A mutants both replicated like the wild type and induced comparable numbers of DMVs in replicon-containing cells, whereas significantly fewer DMVs were observed in the expression system. Although the DMV number in the expression-based system was, in general, 10-fold higher than in replicon-containing cells, variability was also much higher and therefore quantitative information is difficult to gain from this system. Nevertheless, owing to high efficiency of DMV formation, the expression system is ideally suited to obtain qualitative information, especially about mutants that do not support HCV RNA replication.

By investigating the contribution of polyprotein cleavage by NS3-4A on HCV-induced membrane remodeling, we made two important observations. First, in spite of efficient polyprotein trans cleavage by the minimal NS3 protease domain, full-length NS3 was required for DMV formation. Thus, the helicase domain plays an important role in membrane remodeling by the HCV proteins. Whether this is due to structural constraints mediated, e.g., by helicase interaction with other viral or cellular proteins or viral RNA or whether helicase activity per se is needed for membrane remodeling remains to be determined. Second, we found that expression of the viral protease in trans, together with the NS4B-5B polyprotein substrate, induced only very low numbers of DMVs, in spite of highly efficient trans cleavage. Thus, high efficiency of DMV formation requires a contingent NS3-5B polyprotein acting in cis.

Along the same line, we found that regulated polyprotein cleavage kinetics play an important role in DMV formation, as well as RNA replication. HCV polyprotein cleavage occurs in a preferential order, with NS4B-5A representing a rather stable precursor (33) (34) (35) (36) (37) . Interestingly, a mutation previously reported to accelerate cleavage at this site (38) diminished the formation of DMVs, correlating with a reduction of HCV RNA replication. Furthermore, elimination of the NS4B-5A precursor by the insertion of a heterologous IRES between NS4B and NS5A completely blocked DMV biogenesis, arguing that this precursor plays an important role in the formation of the membranous HCV replication factory. The reason why NS4B-5A intermediates are required for DMV formation and RNA replication might be related to a slow process of NS4B or NS5A maturation to allow proper folding, dimerization/oligomerization, or a posttranslational modification such as phosphorylation. In addition, proper folding might play an important role in the interaction of NS5A with host cell factors, e.g., those involved in cellular membrane homeostasis (19, 26) . In this case, the NS4B-5A precursor would exert a spatiotemporal regulation of these interactions. Additionally, features of the NS4B-NS5A precursor itself might play a distinct role in initial steps of HCV membrane-remodeling events. For instance, the linkage of AHs in the NS4B C-terminal domain and at the very N terminus of NS5A in the precursor might exert a membrane activity different from that of the fully processed proteins.

Distinct membrane-remodeling activities of protein precursors have been reported for poliovirus (43) , which, similar to HCV, induces DMVs in infected cells (44) . Moreover, in the severe acute respiratory syndrome coronavirus, three full-length transmembrane-containing nonstructural proteins (nsp3, -4, and -6) are needed to induce the formation of DMV-like structures that are morphologically similar to those found in infected cells (45) . Single expression of each of these nsps leads to a distinct membrane rearrangement such as membrane proliferation or induction of vesicles, but DMV formation appears to require the concerted action of all three nsps (45) . Along these lines, it is also known that the expression of replicase subunits nsp2 to nsp7 of arteriviruses induces membrane changes similar to those in infected cells (46) , with nsp2 and nsp3 playing the main role in inducing a membrane-bound scaffold for the arterivirus replication complex (46) . However, conversely to our findings, cleavage of the nsp2/3 junction by the nsp2 protease was not essential for the formation of DMVs (47) .

In conclusion, we demonstrate that DMVs are induced by a tightly regulated and predominantly cis-acting NS3-5B polyprotein. Key factors revealed in the present study are the NS5A N-terminal AH and D1, as well as regulated polyprotein cleavage in cis by full-length NS3-4A. Deciphering how host cell proteins and lipids are subverted by these viral factors to induce membrane rearrangements, finally leading to the formation of membranous replication factories, will be the next important step.

Plasmid constructs. For descriptions of the plasmids used in this study, see Text S1 in the supplemental material.

Generation and maintenance of cell lines. For information about the cell lines used in this study, see Text S1 in the supplemental material.

In vitro transcription and transfection of cell lines. Synthesis of in vitro transcripts and transfection of replicon RNAs by electroporation have been described in detail elsewhere (48) . pTM vectors allowing the expression of JFH-1-derived polyprotein fragments were transfected with the Mirus TransIT-LT1 Transfection Reagent (Mirus Bio LLC) according to the manufacturer's instructions.

Transient-replication assay. For quantification of the replication efficiency of subgenomic replicons containing a firefly luciferase gene, luciferase activity contained in cell lysates was determined at different time points after electroporation as described elsewhere (49) .

Immunoblot analysis. Western blot analysis was performed as described previously (49) , with antibodies specified in Text S1 in the supplemental material.

High-pressure freezing and freeze substitution. Cells grown on sapphire discs were subjected to high-pressure freezing and subsequent epon embedding as previously described (4) . EM analyses of the embedded cells and quantifications were performed as described in Text S1 in the supplemental material.

CLEM. Huh7-Lunet/T7 cells grown on patterned sapphire discs and transfected with pTM_NS3-5B_⌬D1-GFP were subjected to CLEM as previously reported (4).

Supplemental material for this article may be found at http://mbio.asm.org/ lookup/suppl/doi:10.1128/mBio.00759-15/-/DCSupplemental.

Text S1, DOCX file, 0.1 MB.

Membranous replication factories induced by plus-strand RNA viruses

Architecture and biogenesis of plusstrand RNA virus replication factories

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex

Identification of the hepatitis C virus RNA replication complex in huh-7 cells harboring subgenomic replicons

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment

Hepatitis C virus RNA replication

Hepatitis C virus proteins: from structure to function

Assembly of infectious hepatitis C virus particles

Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B

Amino-terminal amphipathic alpha-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production

Amphipathic {alpha}-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B

NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes

Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A

The NS5A protein of hepatitis C virus is a zinc metalloprotein

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly

Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles

Regulation of hepatitis C virion production via phosphorylation of the NS5A protein

Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A

Hepatitis C virus RNA replication and assembly: living on the fat of the land

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

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

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions

Expression and identification of hepatitis C virus polyprotein cleavage products

Generation and characterization of a hepatitis C virus NS3 protease-dependent bovine viral diarrhea virus

Kinetic and structural analyses of hepatitis C virus polyprotein processing

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

Hepatitis C virus NS3 serine proteinase: trans cleavage requirements and processing kinetics

Characterization of cell lines carrying self-replicating hepatitis C virus RNAs

Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus

Hepatitis C virus nonstructural protein 5A (NS5A) is a RNA-binding protein

Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain

Efficient initiation of HCV RNA replication in cell culture

Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations

Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells

Complex dynamic development of poliovirus membranous replication complexes

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex

Formation of the arterivirus replication/ transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes

Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant

Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication

We thank the Electron Microscopy Core Facilities at Bioquant (University of Heidelberg) and the European Molecular Biology Laboratory (EMBL, Heidelberg) for providing access to their equipment and for excellent support. We especially appreciate the expert technical assistance provided by Andrea Hellwig and Stefan Hillmer (EMCF, University of Heidelberg) and by Rachel Santarella-Mellwig, Charlotta Funaya, and Pedro Machado (EMCF, EMBL, Heidelberg). Special thanks to our colleagues Ulrike Herian, for assistance with cell culture, and Ji-Young Lee, for helpful discussions.This work was supported by grants from the Deutsche Forschungsgemeinschaft (TRR83, TP 13 and SFB 1129, TP 11).