FRET analyses reveal oligomerization of HCV NS4B. (A) Subcellular localization of HCV NS4B and DV NS4B fusion proteins. Constructs pCMVYFP-NS4B (Y-4B), pCMVNS4B-YFP (4B-Y), and pCMVDV4B-YFP (DV4B-Y) were transfected into U-2 OS cells, followed by fixation with 2% paraformaldehyde and confocal laser scanning microscopy, as described in Materials and Methods. (B) Colocalization of HCV NS4B and DV NS4B. Constructs pCMVDV4B-YFP (DV4B-Y) and pCMVNS4B-CFP (4B-C) were cotransfected into U-2 OS cells, followed by fixation with 2% paraformaldehyde. Since the CFP emission was not sufficiently bright for optimal colocalization studies, HCV NS4B in construct 4B-C was detected with polyclonal antibody 86 and Alexa 594-conjugated secondary antibody, as described in Materials and Methods. Confocal laser scanning microscopy revealed colocalization of DV4B-Y and 4B-C. (C) FRET analyses. Constructs pCMVCFP-NS4B (C-4B), pCMVYFP-NS4B (Y-4B), pCMVNS4B-YFP (4B-Y), pCMVNS4B- CFP (4B-C), pCMVDV4B-YFP (DV4B-Y), and pCMVDV4B-CFP (DV4B-C) were transfected into U-2 OS cells in different combinations, as indicated, followed by acceptor photobleaching FRET analyses as described in Materials and Methods. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls for FRET, respectively. Box-and-whisker plots represent the median FRETeff values of 20 measurements (middle line), the FRETeff values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P < 0.0001).

Confirmation of FRET results by coimmunoprecipitation. (A) Constructs pCMVNS4B-HA, pCMVNS4B-FLAG, pCMVDV4B-HA, and pCMVDV4B-FLAG were transfected into U-2 OS cells as indicated, followed by immunoprecipitation (IP) of cell lysates with anti-FLAG M2 agarose and Western blotting (WB) with an anti-HA antibody, as described in Materials and Methods. Direct Western blotting of the cell lysates by use of anti-FLAG and anti-HA antibodies is shown in the lower panels. Molecular mass markers are indicated on the right. (B) Constructs pCMVNS4B_40-130-HA, pCMVNS4B_40-130-FLAG, pCMVNS4B_130-261-HA, pCMVNS4B_130-261-FLAG, and pCMVDV4B-FLAG were transfected into U-2 OS cells as indicated, followed by immunoprecipitation and Western analyses as described for panel A. Molecular mass markers are indicated on the right.

Subcellular localization of NS4B and NS4B segment 40-130 harboring the AH2mut mutations. Constructs pCMVNS4B-YFP (wt), pCMVNS4B_AH2mut-YFP (AH2mut), pCMVNS4B_40-130-YFP (40-130 wt), and pCMVNS4B_40-130AH2mut-YFP (40-130 AH2mut), illustrated schematically at the top, were transfected into U-2 OS cells, followed by fixation with 2% paraformaldehyde and confocal laser scanning microscopy as described in Materials and Methods.

AH2mut mutations disrupt membranous web formation. Ultrathin sections from UHCVcon-57.3 and UHCVcon-AH2mut cells cultured for 48 h in the absence of tetracycline were examined by electron microscopy. While typical membranous webs were found in 17 of 100 sections from UHCVcon-57.3 cells expressing the wild-type (wt) HCV H77 polyprotein, no membranous web was found in 200 sections from UHCVcon-AH2mut cells expressing the H77 polyprotein harboring the AH2mut alanine substitutions in NS4B.

Several determinants contribute to the oligomerization of HCV NS4B. (A) Subcellular localization of NS4B fragments. A schematic representation of NS4B's known and predicted structural elements as well as the different fragments analyzed is depicted at the top. AH, amphipathic α-helix; TM, transmembrane segment; H, α-helix. Constructs pCMVNS4B-GFP (1-261), pCMVNS4B_1-130-GFP (1-130), pCMVNS4B_40-130-GFP (40-130), pCMVNS4B_61-130-GFP (61-130), pCMVNS4B_1-116-GFP (1-116), and pCMVNS4B_130-261-GFP (130-261) were transfected into U-2 OS cells, followed by fixation with 2% paraformaldehyde and confocal laser scanning microscopy. (B) FRET analyses. Constructs pCMVNS4B_40-130-CFP and pCMVNS4B_40-130-YFP (40-130 + 40-130), pCMVNS4B_130-261-YFP and pCMVNS4B_130-261-CFP (130-261 + 130-261), and pCMVNS4B_40-130-CFP and pCMVNS4B_130-261-YFP (40-130 + 130-261) were cotransfected into U-2 OS cells, followed by acceptor photobleaching FRET analyses. Cotransfection of unfused CFP and YFP (CFP + YFP) served as a negative control. The significance of the observed differences was assessed as described in Materials and Methods (*, P < 0.0001).

Oligomerization of NS4B proteins from different HCV genotypes. (A) Amino acid sequence conservation between NS4B proteins from the HCV H77 consensus (genotype 1a; GenBank accession number AF009606) and JFH-1 (genotype 2a; GenBank accession number AB047639) clones. After Clustal W alignment of the two sequences, a score of 1 was attributed to a conserved position and a score of 0 to a divergent one. The mean score of overlapping 7-amino-acid windows was calculated along the sequence and illustrated as the percent identity for each central position of the window. (B) FRET analyses. Constructs comprising H77- and JFH-1-derived NS4B sequences fused to CFP or YFP were transfected in different combinations, as illustrated. See Materials and Methods for individual plasmid denominations. Cotransfection of unfused CFP and YFP (CFP + YFP) served as a negative control. All combinations tested were significantly different from the negative control (P < 0.0001).

Role of amphipathic α-helix AH2 in NS4B oligomerization. (A) Constructs pCMVNS4B-CFP and pCMVNS4B-YFP (both designated wt) as well as pCMVNS4B_AH2mut-YFP and pCMVNS4B_AH2mut-CFP (both designated AH2mut) were transfected into U-2 OS cells in different combinations, as indicated. (B) Constructs pCMVNS4B_40-130-CFP and pCMVNS4B_40-130-YFP (both designated wt), pCMVNS4B_40-130AH2mut-YFP and pCMVNS4B_40-130AH2mut-CFP (both designated AH2mut), and pCMVNS4B_130-261-YFP (130-261) were transfected into U-2 OS cells in different combinations, as indicated. The significance of the observed differences was assessed as described in Materials and Methods (*, P < 0.0001; #, P = 0.0036).