Figure 1. Rationale for mutagenesis in NS2 transmembrane region.

(A) Schematic representation of the topology of E2, p7 and NS2 proteins. (B) Position of the inserted alanine residues in the putative N-terminal membrane domain of NS2. Alignments of NS2 membrane domain sequences from HCV strains JFH1 (genotype 2a, accession number AB047639) and Con1 (genotype 1b, AJ238799). Amino acids are numbered with respect to NS2 and the HCV JFH1 polyprotein (top row). Second. struct., secondary structure deduced from the NMR analyses of NS2 synthetic peptides from Con1 strain [19] (Jirasko et.al. the 16^th international Conference on HCV and Related Viruses, Nice, October 3–7, 2009); c = coil, h = helix; capital letters indicate canonical helix structure. Predicted TM, consensus transmembrane (TM) segment predictions were deduced from a set of 6 available web-based algorithms prediction methods (DAS, TOPPRED2, TMHMM, SOSUI, TMPRED, PHD-TM) and represented by stretches of “T”. Arrows indicate the positions of the various alanine insertions. (C) Ribbon representations of the molecular homology model of NS2[1–27] of JFH1 (left) and the theoretical model for alanine insertion mutant A16 (right). An Ala insertion (shown in magenta) twists the helix by 110°. The N-terminal part of the model is shown in the same orientation as in the left model to highlight the distortion of residue positions on the C-terminal part of the helix. The side chains of indicated residues are shown to highlight this distortion. These models were constructed by using the NMR structure of Con1 NS2[1–27] ([19]; PDB entry, 2JY0) as template and the Swiss-PdbViewer program (http://www.expasy.ch/swissmod/). Residues are colored based on the chemical properties of their side chains: hydrophobic (gray) and polar (yellow). Acidic (Asp) and basic (Arg, Lys) residues are red and blue, respectively. His is cyan, and Gly is light gray. The membrane interfaces and hydrophobic core are schematically represented.