After 60 min incubation on ice, cell debris was removed by 15 min centrifugation at 20,000xg. into the hydrophobic core of the lipid bilayer are indicated by the dash bars. (C) Far-UV circular dichroism (CD) analyses of synthetic peptides NS2- and NS2[60-99] in various membrane mimetic environments. CD spectra were recorded in either 50% 2,2,2-trifluoroethanol (TFE) or 1% L–lysophosphatidyl choline (LPC), or the following detergents: 100 mM sodium dodecyl sulfate (SDS), 100 mM n-dodecyl–D-maltoside (DM), or 100 mM dodecyl phosphocholine (DPC). (D, E) NMR analysis of the peptides in 50% TFE. A summary of sequential (conversation with the core protein . This conversation appears to be regulated by casein kinase II-mediated phosphorylation of NS5A . Assembly of HCV particles is usually tightly linked to lipid metabolism, LDs and the machinery required for production and secretion of very-low-density lipoproteins (VLDL) , C. Several models of HCV assembly have been put forward, but the precise details are unknown (examined in ). While these models can explain the early actions of nucleocapsid formation, it is unclear how these nucleocapsids acquire the membranous viral envelope and the envelope glycoproteins and how this process is usually linked to VLDL formation and secretion. NS2 may play a central role in these reactions, but the precise mechanisms are not known , . In this study we undertook a detailed structural and functional characterization of the N-terminal MBD of NS2. We solved the NMR-structures of TMS2 and TMS3 and propose a model of NS2 membrane topology. In addition, we performed a structure-activity study of the MBD and established an conversation map of NS2. HS-1371 The data reveal that NS2 serves as a key organizer participating in multiple protein-protein interactions that are required for the assembly of infectious HCV particles. Results Mapping of transmembrane domains in NS2 and tentative model of its membrane topology We reported recently that a transmembrane segment denoted TMS1 was almost invariably predicted in the very N-terminal region (aa 1C23) of NS2, irrespective of the analyzed genotypes and subtypes . TMS in the 23C102 region ( HS-1371 and recommendations therein) yielded inconsistent results that depended both around the genotype examined and the method used (data not shown). By using secondary structure predictions and the algorithm developed by Wimley and White to calculate the propensity of an aa sequence to interact with membranes (Physique S1, A and B) we could deduce that this consensus segments 17C45 and 72C96 exhibit a clear propensity to partition into the membrane bilayer and likely include transmembrane helical passages (Physique 1A and supplementary Physique S1). In contrast, the aa segment 49C71 is predicted not to HS-1371 show such properties. Based on these results, the NS2 MBD sequence was divided into the three segments: 1C27, 27C59, and 60C99, each made up of a putative transmembrane helix (Physique 1A). Open in a separate window Physique 1 Model of the membrane binding domain name of NS2 and its orientation in the membrane.(A) Sequence comparison of the NS2 segment 1C100 from Con1 and JFH1 used to design the NS2 mutants. Amino acids are numbered with respect to NS2. The helical segments in the membrane binding domain name deduced from NMR analyses of Con1 NS2 peptides ( and this study) are shown at the (h, helical). Identical, highly similar, and comparable residues at each position are symbolized by an and methylation site affecting the BspEI cleavage site at this position. To generate genomes with double tagged NS2, oligonucleotides encoding the Flag-, or hexahistidine- or HA-tag fused to the GSG linker were inserted in-frame into the BspEI site. transcription and electroporation of HCV RNAs The experimental procedures used to generate in vitro transcripts from cloned HS-1371 HCV sequences and transfection of Huh-7 cells by electroporation have been described in detail recently . For trans-complementation assays a mixture of 7.5 g NS2 mutant and 5 g helper replicon RNA was used. After electroporation, cells were immediately transferred to STAT6 total DMEM and seeded as required for the assay. Immunohistochemical staining.