Amino acid sequence analyses and secondary-structure predictions of the N-terminal segments of NS5A proteins from HCV and related viruses. Sequence analyses and amino acid repertoires of the N-terminal segments of NS5A from (A) HCV, (B) GBV-B, (C) GBV-A, (D) GBV-C, (E) BVDV, and (F) CSFV are shown on the left. The HCV NS5A sequence was used as a reference for amino acid position numbering. The sequence variability observed in natural variants for each virus is presented as the repertoire of amino acids at each position, in decreasing order of observed frequency, from top to bottom. Repertoires were deduced from multiple-sequence alignments, including 116, 3, 3, 15, 16, and 8 sequences for HCV, GBV-B, GBV-A, GBV-C, BVDV, and CSFV, respectively. The least frequently observed residues at each position were not reported, i.e., less than two times for GBV-C, BVDV, and CSFV and less than 2% for HCV, as they might have been due to PCR and/or sequencing errors and/or sequencing of defective viruses. However, all variations were considered for GBV-B and GBV-A, since only three sequences were reported for each of these viruses. Hydrophobic (F, I, W, Y, L, V, M, P, C, A, and G) and hydrophilic (T, S, K, Q, N, H, E, D, and R) amino acids are color coded in black and pink, respectively (classification based on the Eisenberg hydrophobicity scale [18]). The hydropathic consensus patterns deduced from repertoires are letter coded as follows: i, hydrophilic positions; o, hydrophobic positions; v, variable positions (i.e., positions where both hydrophilic or hydrophobic residues were observed). Conserved hydrophilic and hydrophobic positions in all virus sequences are in boldface characters and are highlighted in yellow. Above each repertoire, experimentally determined (46, 50) or predicted alpha helices are shown in blue and gray, respectively. The corresponding helical-wheel projections are shown on the right. Note that only the helical segments common to all viruses and including the conserved hydrophilic and hydrophobic positions were reported. The corresponding segment and the SwissProt-TrEMBL ID are indicated for each virus prototype sequence.

The N-terminal 27 to 33 amino acids of GBV-B, GBV-C, and BVDV NS5A proteins are sufficient to mediate membrane association. (A) Amino acid sequences of the GBV-B (Q6QLR5), GBV-C (Q96899), and BVDV (P19711) isolates used in this study. (B) The N-terminal 27 to 33 amino acids of GBV-B, GBV-C, and BVDV NS5A proteins were fused to either the N or C terminus of GFP. (C) GFP fusion constructs were expressed in U-2 OS cells and examined by confocal laser scanning microscopy, as described in Materials and Methods.

Subcellular distributions of full-length and N-terminally truncated NS5A proteins from GBV-B, GBV-C, and BVDV. U-2 OS cells were transfected with constructs pCMV-myc-GBV-B-5A-HA (GBV-B-5A), pCMV-GBV-B-5AΔN33-HA (GBV-B-5AΔN33), pCMV-GBV-C-5A-FLAG (GBV-C-5A), and pCMV-GBV-C-5AΔN27-FLAG (GBV-C-5AΔN27) and fixed 48 h later. Pools of cell lines inducibly expressing constructs pUHD-BVDV-5A-HA (BVDV-5A) and pUHD-BVDV-5AΔN28-HA (BVDV-5AΔN28) were analyzed 48 h following tetracycline withdrawal. Proteins were visualized with a polyclonal anti-HA antibody or MAb 9E10 directed against the c-myc epitope tag, followed by detection with secondary antibodies, as described in Materials and Methods.

Chimeric HCV replicons. The highly conserved 6-amino-acid stretch in the N-terminal portion of the amphipathic alpha helices (Fig. 1) is boxed. Substituted amino acid residues are highlighted in gray.

CD analyses of synthetic peptides GBV-C NS5A_1-27 and BVDV NS5A_1-28. CD spectra of (A and B) GBV-C NS5A_1-27 and (C and D) BVDV NS5A_1-28 were recorded in water (pH 4.0) or 10 mM sodium phosphate buffer, pH 7.0, complemented with either 50% TFE (solid line) in panels A and C or the following detergents in panels B and D: 100 mM SDS (dashed line), 100 mM dodecyl phosphocholine (DPC) (large dashed line), and 100 mM n-dodecyl β-d-maltoside (DM) (dotted line). deg, degrees.

Membrane flotation analyses of GFP fusion constructs. (A) Schematic diagram of the membrane flotation procedure. Following hypotonic lysis, postnuclear lysates were loaded at the bottom of a 37.5 to 5% Nycodenz gradient. During equilibrium centrifugation for 20 h at 100,000 × g, membranes float to the upper, low-density fractions. Seven fractions of equal volume were collected from the top and analyzed by immunoblotting. (B) Lysates of U-2 OS cells transfected with pCMV-GBV-B-5AN33-GFP, pCMV-GBV-C-5AN27-GFP, and pCMV-BVDV-5AN28-GFP were analyzed. p63 was included as a control. Flotation of this integral ER membrane protein was abolished by treatment of the membranes with 1% Triton X-100 for 20 min at 4°C. MAbs JL-8 against GFP and G1/296 against p63 were used for immunoblotting.

Membrane flotation analyses of full-length and N-terminally truncated NS5A proteins. U-2 OS cells were transfected with pCMV-GBV-B-5A-HA (GBV-B-5A), pCMV-GBV-B-5AΔN33- HA (GBV-B-5AΔN33), pCMV-GBV-C-5A-FLAG (GBV-C-5A), and pCMV-GBV-C-5AΔN27-FLAG (GBV-C-5AΔN27), followed by hypotonic lysis and membrane flotation analyses 48 h posttransfection. Cell pools stably expressing pUHD-BVDV-5A-HA (BVDV-5A) or pUHD-BVDV-5AΔN28-HA (BVDV-5AΔN28) were derepressed by tetracycline withdrawal for 48 h. A horseradish peroxidase-labeled anti-HA antibody and MAb M2 against the FLAG tag were used for immunoblotting.

RNA replication, polyprotein processing and subcellular localization of chimeric constructs. (A) RNA was in vitro transcribed from the indicated replicon constructs and electroporated into Huh-7.5 cells, followed by plating on 100-mm-diameter dishes at 6 × 10⁵, 6 × 10⁴, and 6 × 10³ cells per dish and G418 selection, as described in Materials and Methods. G418-resistant colonies were stained with crystal violet after 3 weeks. GBV-C 4-29 is representative of all other constructs, as well as the control construct pCon1/SG-Neo(pol⁻)/GFP-FLAGI.1, which did not yield any viable clones. wt, wild type. (B) Chimeric constructs in the context of the entire nonstructural-protein region were expressed from a T7 promoter-driven expression construct in Huh7-T7-IZ cells. Cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis, followed by immunoblotting using MAb 11H against HCV NS5A. (C) Huh7-T7-IZ cells transfected as in panel B were analyzed by immunofluorescence microscopy using MAb 11H.