Influence of mutations in the NS4B C-terminal region on production of infectious virus. (A) Schematic representation of the full-length JFH1 genome and the JFH1ΔE1E2 mutant containing a deletion removing most of the envelope glycoprotein coding region (codons 217 to 567 [50]). The asterisk indicates the NS4B region into which the mutations were introduced. (B) Release kinetics of infectious HCV particles. Huh7-Lunet cells were transfected with the HCV RNA specified at the bottom of the panel. Culture supernatants were harvested at given time points, and infectivity titers were determined by limiting-dilution assay and are expressed as TCID₅₀/ml. The mean values of two independent experiments are shown; error bars indicate standard deviations. (C) Efficiency of virus assembly and release. Huh7-Lunet cells were transfected with given full-length HCV RNAs. After 48 h, the cells and supernatants were harvested and subjected to three freeze-thaw cycles, and the infectivity titers contained in the cell lysates (intracellular) and corresponding supernatants (extracellular) were determined by limiting-dilution assay. The mean values of two independent experiments are shown; error bars indicate standard deviations. (D) Expression levels of NS5A at 72 h posttransfection. Arrows indicate the basal (p56) and hyperphosphorylated (p58) forms of NS5A, respectively. Bands were quantified by densitometry, and the relative protein levels normalized to the wt are indicated at the bottom of the panel as percentages.

Pseudoreversions rescuing the replication defect of NS4B C-terminal mutants. (A) Schematic representation of primary NS4B mutations and the corresponding pseudoreversions. The NS4B C-terminal half (aa 130 to 261) containing TM3+TM4, as well as cytoplasmic helices H1 and H2, is depicted. The structure of the NS5A dimer (48) with the N-terminal amphipathic helix attached to a membrane bilayer is indicated. Primary mutations and corresponding pseudoreversions are labeled with the same color. Arrows originate from the primary mutation and point toward the corresponding pseudoreversion. Solid lines indicate functional rescue; broken lines indicate there was no such rescue. (B to H) Huh7-Lunet cells were transfected with luciferase reporter replicons specified in the right of each panel. Cells were lysed 4, 24, 48, and 72 h after transfection, and the luciferase activity in cell lysates was determined. The data were normalized to the 4-h value that reflects the transfection efficiency. Mean values from two independent experiments are shown; error bars indicate the standard deviations. (B) NS4B mutant F211A and pseudoreversion L208F in NS4B. (C) NS4B mutant F211A and pseudoreversions T221N in NS4B and K139E in NS5A. (D) NS4B mutant R214A and pseudoreversion R192G in NS4B. (E) NS4B mutant P220A and pseudoreversion G200E in NS4B. (F) NS4B mutant P220A and pseudoreversion I253M in NS4B. (G) NS4B mutant E226A and pseudoreversion G200S in NS4B. (H) NS4B mutant E226A and pseudoreversions A201E in NS4B and the intergenic compensatory mutation K139E in NS5A.

Mutations in the C-terminal region of NS4B and their impact on interaction with the NS4B N-terminal region. (A) Heterotypic interaction between the N-terminal and the C-terminal NS4B fragment. U2-OS cells were transfected for 24 h with expression plasmids encoding for NS4B CFP and YFP fusion proteins depicted in the lower right prior to FRET acceptor photobleaching. The C-terminal NS4B fragment corresponds either to the wt sequence or contains a mutation (▴) specified in the bottom of the diagram. Coexpression of CFP and YFP (CFP+YFP) and expression of a CFP-YFP fusion protein (CFP-YFP) served as negative and positive controls, respectively. Box-and-whisker diagrams show the median FRET efficiency (middle line) from 32 measurements. The bottom and top lines in the boxes represent the 25th and 75th percentiles, respectively. Minimum and maximum values are indicated by vertical lines. The dashed line indicates the background, as determined by the negative control. (B) Analogous to panel A but showing a larger panel of primary NS4B mutants. (C to F) Pseudoreversions restoring heterotypic NS4B interaction. U2-OS cells were transfected with constructs as described in the text, and interaction was determined by FRET analysis. The results are shown for mutants F211A (C), R214A (D), P220A (E), and E226A (F). For each data set, the statistical significance was calculated by using the unpaired Student t test as described in Materials and Methods (*, P < 0.0001; †, P = 0.0017).

Effects of mutations in the C-terminal NS4B region and pseudoreversions on HCV-induced membrane rearrangements. Lunet-T7 cells were transfected with the NS3-5B expression vector containing either wt NS4B or given mutation(s). After 24 h, the cells were fixed, processed, and analyzed by TEM as described in Materials and Methods. (A) DMV morphology induced by replication-competent NS3 to NS5B containing wt NS4B (i) or the replication-competent NS4B mutants E199A (ii) or H217V (iii). (B to E) DMV morphology induced by replication-incompetent NS4B mutants specified in the left of each panel (i) and the corresponding pseudorevertants (subpanels ii and iii in each panel). Regular (*) and aberrant (▾) DMVs are indicated. The scale bar in each panel represents 100 nm.

Residues in the C-terminal domain of NS4B are important for HCV RNA replication. (A) Predicted NS4B membrane topology. The schematic representation shows the N-terminal amphipathic α-helices AH1 and AH2, the four transmembrane segments (TM1 to TM4), the nucleotide binding motif (NBM), the C-terminal α-helices H1 and H2, and the very C-terminal palmitoylation site(s). The reported posttranslational flip of the N terminus into the ER lumen is indicated by the arrow. (B) HCV JFH1 protein sequence (2a_JFH) of the NS4B C-terminal region. NS4B amino acid (aa) positions are given. Fully conserved amino acids are marked with asterisks. Alternative amino acid residues found in other genotypes are indicated below. Residues targeted in this mutagenesis study are underlined and highlighted with bold letters. (C) Schematic representation of the subgenomic luciferase reporter replicon. The luciferase is expressed as an N-terminal fusion with 16 aa of the N-terminal region of the core protein (black line) and is translated under the control of the HCV IRES contained in the 5′UTR. The second cistron (NS3 to NS5B) is translated via the internal ribosome entry site of the encephalomyocarditis virus (E-IRES). The star indicates the region in NS4B targeted by the mutations. (D) Huh7 cells were transfected with in vitro transcribed luciferase replicon RNAs specified in the bottom. Cells were lysed 4, 24, 48, and 72 h after transfection, and the luciferase activity in cell lysates was determined. The data were normalized to the 4-h value that reflects transfection efficiency. The background of the assay is determined with the active-site NS5B polymerase mutant (ΔGDD). The mean values of two independent experiments are shown. Error bars indicate standard deviations.

Identification of pseudoreversions compensating for NS4B defects. (A) Schematic representation of the selectable subgenomic replicon. The neomycin phosphotransferase is expressed as an N-terminal fusion with 16 aa of the N-terminal region of the core protein (black line) and is translated under the control of the HCV IRES contained in the 5′UTR. The second cistron (NS3 to NS5B) is translated via the internal ribosome entry site of the encephalomyocarditis virus (E-IRES). The asterisk highlights the region into which the NS4B mutations were introduced. (B) Experimental approach used to select for pseudoreversions. Huh7 cells were transfected with selectable replicon RNAs, and cells were cultured in the presence of 250 or 500 μg of G418/ml for 4 weeks. For each mutant two cell clones were expanded, the total RNA was isolated, and at least two cDNA clones per cell clone were sequenced. (C) Number of G418-resistant cell colonies. Arrows indicate those mutants for which no colonies could be selected.

Impact of mutations in the C-terminal region on NS4B protein stability. Huh7-Lunet T7 cells were transfected with the expression vector (control) or the same vector encoding the NS3-5B polyprotein fragment corresponding to wt NS4B or a NS4B mutant specified in the top. Cells were harvested 24 h posttransfection and subjected to Western blot analysis by using monospecific antibodies for NS4B or NS5A. The numbers below each lane refer to the relative amounts of NS4B proteins as determined by densitometry. After subtraction of the background, the NS4B signals were normalized to those of the corresponding NS5A. One result of two independent experiments is shown.

Ultrastructural analysis of membrane rearrangements induced by the HCV replicase proteins. (A) Huh7-Lunet cells were transfected with in vitro transcribed subgenomic replicon RNA. After 24 h, the cells were fixed, processed, and analyzed by TEM as described in Materials and Methods. The boxed area in the left panel is shown enlarged in the right panel. (B) Huh7-Lunet T7 cells were transfected with the expression vector encoding nonstructural proteins NS3 to NS5B (i and ii) or empty vector (iii). After 24 h, the cells were fixed, processed, and analyzed by TEM. The boxed area in the left panel (i) is shown enlarged in the middle panel (ii). (C) Time-dependent membrane alterations induced by expression of the HCV replicase proteins (NS3 to NS5B). Huh7-Lunet T7 cells were fixed 8 h (i), 16 h (ii), and 24 h (iii) after transfection and analyzed by TEM. The boxed areas in the middle and right panels are shown enlarged below. (D) Percentage of cells displaying membrane rearrangements and/or DMVs at given time points after transfection. Note the time-dependent increase of DMVs. For each time point, ∼30 cells were examined. White scale bar, 1 μm; black scale bar, 100 nm. AV, autophagic vacuole; DMVs, double membrane vesicles; ER, endoplasmic reticulum; LD, lipid droplet; M, mitochondrium; MVB, multivesicular body; N, nucleus.

Quantitative analysis of DMV morphotypes. At least 100 vesicular structures per sample from at least three different cells were counted. For each construct the ratio of regular DMVs per total HCV-induced vesicular structures (regular DMVs, aberrant DMVs, and MMVs) is given. The significance of the differences was calculated by using the two-sided Fisher's exact test and is highlighted for the wt and the primary mutants (dashed lines) or the primary mutant and corresponding pseudorevertants (black lines) (*, P < 0.0001; †, P < 0.01).