A Deletion in NS5A Domain III Disrupts the Production of Infectious Virus.

<p>A). Schematic representation of the domain organization of NS5A indicating the locations of the three domains and low complexity domain connectors (LCS). The location of the membrane anchoring helix is also shown. Numbers indicate amino acid positions on the Con1 genotype 1b NS5A sequence. Sequences of the C terminus of NS5A indicated by the black box on the schematic are shown below for representative sequences of major HCV genotypes. The sequences shown are 1a H77 (AF011753), 1b Con1 (AJ238799), 2a JFH-1 (AB047639), 2b HC-J8 (D10988), 3a HCVCEN (X76918), 4a ED43 (Y11604), 5a EUH1480 (Y13184), and 6a 6a33 (AY859526). B). IHC analysis of cells electroporated with indicated viral RNAs (top of image rows) for NS5A 48 hours post electroporation (top row of panels) or 48 hours post infection (bottom row of panels). C). Western blot of wild type (WT) and deletion B (del B) NS5A from cells electroporated with replication competent full-length genomes shown in B and D. D). RNA replication 48 hour time course of J6/JFH-1 (blue line), J6/JFH-1 deletion B (red line), or J6/JFH-1 pol- (black line) as determined by real time PCR analysis. E). RNA replication 48 hour time course of indicated subgenomic replicon RNAs. pSGR-JFH-1 (red line), pSGR-JFH-1 deletion B (blue line), and pSGR-JFH-1 pol- (black line). F). Infectivity release 48 hours (gray bars) and 6 days post (black bars) electroporation from indicated viral RNA electroporated cells in units of tissue culture infectious dose 50% value per milliliter. G). Infectivity released from cells via freeze-thaw treatment 48 hours post electroporation from indicated viral RNA electroporated cells. H). Relative HCV RNA release from cells electroporated with the indicated HCV constructs. Values reported are arbitrary units normalized to the RNA release by a non-replicating J6/JFH-1 pol-.