Genome replication phenotypes of NS5A domain I mutants in Huh7 and Huh7.5 cells.

<p><i>In vitro</i> transcripts of mSGR-luc-JFH-1 containing the indicated mutations were electroporated into either Huh7 (<b>A</b>) or Huh7.5 (<b>B</b>) cells. Luciferase activity was measured at 4, 24, 48 and 72 h post-electroporation (h.p.e.) and was normalized to 4 h.p.e. Data from three independent experiments are shown and error bars represent the standard error of the mean. ns: no statistically significant difference from WT.</p

Quantification of the effect of the V67A and P145A mutations on the size of LD.

<p><b>A</b> LDs in Huh7.5 cells electroporated with the indicated JFH-1 constructs were visualized by staining with BODIPY 558/568-C₁₂. <b>B</b> The size of individual LD was determined and plotted as a histogram. The area (μm²) is taken as an indication of the three-dimensional volume of the LD. For comparison similar data was determined from uninfected Huh7.5 cells.</p

V67A and P145A disrupted the co-localization between NS5A and Core or LDs.

<p><b>A</b> Quantification of the percentages of NS5A colocalized with LD (white blocks), or LD colocalised with NS5A (red blocks). <b>B</b> Quantification of the percentages of NS5A colocalized with Core (white blocks), or Core colocalised with NS5A (green blocks). <b>C</b> Quantification of the percentages of Core colocalized with LD (green blocks), or LD colocalised with Core (red blocks). <b>D</b> Spatial data for the distance of LDs from the nuclear envelope were determined from 10 cells for each construct using Fiji. **** indicates significant difference (P<0.0001) from the results for WT.</p

V67A and P145A disrupt the recruitment of NS5A and Core to LDs.

<p><b>A</b> Western blot analysis of NS5A and Core proteins, the LD marker protein ADRP and GAPDH in purified LD fractions compared with whole cytoplasm, cytoplasmic membrane and cytosolic fractions. The abundance of NS5A (<b>B</b>) and Core (<b>C</b>) in the LD fractions was quantified and normalised to the LD fraction ADRP value. <b>D</b> Amount of viral RNA in LD fractions was determined by qRT-PCR. Error bars represent the standard error of the mean of three independent experiments. ** indicates significant difference (P<0.01) from WT.</p

Residues at positions V67 and P145 of domain I are involved in NS5A dimerization.

<p><b>A</b> Input of His-SUMO-domain I (35–215) (left), GST control protein and GST-domain I (35–215) (right), analysed by Western blotting using either anti-His or anti-GST antibodies. <b>B</b> His-tagged domain I proteins were also used as prey in pulldown assays with GST or GST-Domain I with corresponding mutations as bait. Precipitated proteins were analysed by Western blotting using anti-His and anti-GST antibodies. The His:GST ratio was calculated following quantification of Western blot signals using a Li-Cor Odyssey Sa infrared imaging system and represented graphically as a measure of the dimerization activity. These data were representative of three independent experiments using different batches of purified domain I proteins. ** indicates significant difference (P<0.01) from WT.</p

Mutations in NS5A domain I disrupt the production of infectious virus.

<p><i>In vitro</i> transcripts of mJFH-1 containing the indicated mutations were electroporated into either Huh7 (<b>A, B</b>), or Huh7.5 (<b>C-E</b>) cells. Virus genome replication and protein expression was assayed by quantification of NS5A positive cells 48 h.p.e. for Huh7 (<b>A</b>) or Huh7.5 (<b>C</b>) cells by using the Incucyte-ZOOM [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006834#ppat.1006834.ref062" target="_blank">62</a>]. (<b>B</b>, <b>D</b>) Intracellular and extracellular infectious virus was titrated at 72 h.p.e. <b>E</b> Huh7.5 cell lysates at 72 h.p.e. were analysed by western blot with anti-NS5A, anti-Core and anti-β-actin antibodies. Data from three independent experiments are shown and error bars represent the standard error of the mean.</p

Time-course immunofluorescence analysis of LDs, NS5A and Core in WT infected cells.

<p>Huh7.5 cells were electroporated with an <i>in vitro</i> transcript of mJFH-1 WT. At the indicated h.p.e. cells were fixed and stained with anti-NS5A and Core antibodies, BODIPY 558/568-C_12, and DAPI and imaged by Airyscan microscopy (<b>A</b>). Spatial data for LDs were determined from 10 cells for each time point using Fuji. These data were used to determine the number of LDs per cell (<b>B</b>), the average size of LDs (<b>C</b>) and the distance of each LD from nucleus at different time points (<b>D</b>). **** indicates significant difference (P<0.0001) from the results for LDs in untransfected cells. The scale bars are 5μm and 0.5 μm, respectively.</p

Co-localisation of NS5A, Core and NS3 in infected cells.

<p>Huh7.5 cells were electroporated with <i>in vitro</i> transcripts of mJFH-1 WT or the indicated mutants. At 72 h.p.e. cells were fixed and stained with anti-NS5A, NS3 and Core antibodies, and counterstained with DAPI, prior to imaging by Airyscan microscopy. The scale bars are 5 μm and 0.5 μm, respectively.</p

Density gradient analysis of mutant viruses.

<p>Huh7.5 cells were electroporated with <i>in vitro</i> transcripts of WT or the indicated virus mutants. Concentrated culture medium was fractionated using 10–40% iodixanol density-gradient centrifugation. For each fraction, HCV RNA (<b>A</b>) and infectivity (<b>B</b>) were plotted against the buoyant density (n = 3), and Core protein in each fraction was detected by western blot (C). 1 to 12 in (C) indicated the fractions collected from top to bottom with the buoyant density indicated in (A) and (<b>B</b>). The result of a representative of three independent experiments is shown.</p

Residues at positions V67 and P145 of domain I are involved in NS5A RNA binding.

<p><b>A</b> Representative slot blot analysis of RNA-protein complexes captured on nitrocellulose membrane in a filter binding assay using increased amounts of purified His-tagged NS5A domain I (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006834#ppat.1006834.s006" target="_blank">S6 Fig</a>), and a constant amount of ³²P-labelled HCV 3’UTR (or control RNA [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006834#ppat.1006834.ref032" target="_blank">32</a>]). % RNA bound is shown graphically, quantified by phosphoimaging analysis. <b>B</b> Huh7.5 cells were electroporated with <i>in vitro</i> transcripts of mJFH-1 WT or the indicated mutants. Cells were lysed at 72 h.p.e. and NS5A was immunoprecipitated from cell lysates. After washing the beads were subjected to analysis by Western blot and RNA extraction. qRT-PCR were performed to quantify the level of (+) genome RNA bound to NS5A. The graph on the right shows the ratio of RNA copies to NS5A (n = 2). <b>C</b> As <b>B</b> but in this case Core was immunoprecipitated using a rabbit polyclonal anti-Core antibody. ** indicates significant difference (P<0.01) from WT.</p