Genome scale functional profile of HCV.
Graphical representation of the location and phenotype of 15-nt insertions in the HCV genome are shown. For each 15-nt insertion mutant, the ratio of peak area was calculated between selected and non-selected pools and plotted in a bar graph. The lethal phenotype (critical region, red bar) is an absence of an insertion mutant in the selected population. The attenuated phenotype (less critical region, blue bar) is an over two-fold reduction in replication. The tolerated phenotype (dispensable region, green bar) is replication competent. (A) The final assembly shows the fold change (log10) and locations of insertions in the HCV genome. A cartoon of the HCV regions is aligned at the top of graph to show the boundary of each region. The numbering corresponds to the nucleotide (nt) position of JFH-1 genome. (B) The location and phenotype of insertions at the NS5A region are shown. A schematic diagram of the NS5A domains is aligned with the functional profile graph. Note that many insertions at domain 3 are tolerated. (C) The crystal structure of NS5B  (PDB accession code 1C2P), RNA dependent RNA polymerase, displays the functional profiling phenotypes. The front and back views of ribbon and surface diagrams of the NS5B monomer is shown. The fingers, thumb, and palm sub-domains are indicated. The amino acid residues are color coded for insertion phenotypes: red (lethal), blue (attenuating), green (tolerated), and grey (no insertion). Insertions in the sub-domains forming the catalytic active site were lethal (front view) for virus replication, whereas many insertions on the outer surface (back view) were tolerated. The crystal structure was analyzed using PyMOL Viewer. aa, amino acid.