This analysis revealed that for both V67A and P145A there was a significant reduction in genomic RNA associated with LDs (Fig 9D), consistent with a scenario whereby NS5A transports nascent genomes to LDs where it is transferred to the Core protein for subsequent movement to assembly sites. Open in a separate window Fig 9 V67A and P145A disrupt the recruitment of NS5A and Core to LDs. A 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. electroporated into either Huh7 (A) or Huh7.5 (B) cells. Luciferase activity was measured at 4, 24, 48 and 72 h post-electroporation (h.p.e.) and plotted as absolute values. 4 h.p.e. values are indicative of input translation and reflect transfection efficiency. Data from three independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s002.tif (12M) GUID:?36E1E99D-D384-4D9E-B100-40AE18884AB4 S3 Fig: Comparison of replication of NS5A mutants in Huh7 and Huh7.5 cells and analysis of polyprotein processing. A. WT represents the wild type mSGR-luc-JFH-1. P35A, V67A, and P145A are the mutants of domain I which can replicate at lower levels than WT in Huh7 cells; D329 is located at the C terminus of NS5A domain II. The graph shows the RLU values at 72 h.p.e. expressed as a fold increase over the 4 h.p.e. values. B. Huh7.5 cells were transfected with pCMV10-NS3-NS5B expression vectors containing the corresponding mutations. At 48 h.p.t., cell lysates were harvested in GLB and analysed by SDS-PAGE and Western blotting with anti-NS5A (sheep) and anti-NS3 (mouse). The ratio of NS5A:NS3 was calculated following quantification of Western blot signals using a Li-Cor Odyssey Sa infrared imaging system. Data from three independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s003.tif (10M) GUID:?0AF75969-47C0-41A2-97AA-696D2E953033 S4 Fig: Incucyte ZOOM visualisation of virus replication and infection. Indirect immunofluorescence analysis for NS5A expression in Huh7.5 cells electroporated with the indicated viral RNAs at 48 h.p.e. (top row). The middle row shows NS5A expression in cells infected with culture supernatants harvested from the cells presented in the top row. Infected cells were analysed at 48 h.p.i. The bottom row shows NS5A expression at 48 h.p.i. in cells infected with cell lysates from the cells in the top rowCthis represents intracellular virus. After fixation, cells were stained with NS5A antibody and then with Alexa Fluor 568-conjugated donkey anti-sheep IgG (red fluorescence).(TIF) ppat.1006834.s004.tif (5.5M) GUID:?589A4919-8F80-4256-9F39-8A9733A45C03 S5 Fig: Revertant and trans-complementation analysis of the phenotype of V67A and P145A in virus assembly. A. Phenotypes of V67A and P145A are not derived from acquisition of an additional compensatory mutation during the cloning process. Revertants were generated by cloning a WT NS5A fragment back into the mJFH-1 V67A or P145A mutant plasmids. Huh7.5 cells were electroporated with in vitro transcripts of the resulting V67 or P145 revertants. Virus genome replication and protein expression was assayed by quantification of NS5A positive cells 48 h.p.e. by using the Incucyte-ZOOM [62]. Intracellular and extracellular infectious virus was titrated at 72 h.p.e. B. In vitro transcribed WT JFH-1 or the indicated mutant RNAs were co-electroporated with the helper RNA (mSGR-Luc-JFH1) into Huh7.5 cells. 72 h.p.e., supernatant was harvested and cells were lysed by repetitive freeze-thaw cycles. Extracellular and intracellular virus was then titrated in Huh7.5 cells and viral infectivity was determined by using Incucyte ZOOM at 48h.p.i. Data from two independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s005.tif (11M) GUID:?12C502FF-D414-44EC-8329-90C92EAFA674 S6 Fig: A. Time-course immunofluorescence analysis of LD in HCV infected cells. Huh7 cells were infected with mJFH-1 WT at an M.O.I. of 0.5 ffu/cell. At the indicated h.p.e. cells were fixed and stained with BODIPY 558/568-C12, and DAPI and imaged by Airyscan microscopy. B. Colocalisation of NS5A and Rabbit polyclonal to KIAA0174 NS3. Quantification of the percentages of NS5A colocalized with NS3 (white blocks), or NS3 colocalised with NS5A (red blocks) as shown in Fig 8. Co-localisation calculations were performed on 5 cells from at least two independent experiments.(TIF) ppat.1006834.s006.tif (14M) GUID:?4977CFBD-4E61-41F9-8291-FA5CB598B7CF S7 Fig: Expression of WT and domain I mutants for RNA filter.WT represents the wild type mSGR-luc-JFH-1. in the JFH-1 sequence, compared to consensus, particularly the 18 amino acid insertion between residues 432C450. C. Analysis of the three dimensional structures of domain I (1ZH1 and 3FQM) using Pymol. Residues highlighted are the conserved amino acids that are located on the surface of two dimeric conformations at positions indicated in S1 Table.(TIF) ppat.1006834.s001.tif (14M) GUID:?E6FB2EB1-37B6-49AF-8B68-7E2B7625F21C S2 Fig: Genome replication of NS5A domain I mutants. transcripts of mSGR-luc-JFH-1 containing the indicated mutations were electroporated into either Huh7 (A) or Huh7.5 (B) cells. Luciferase activity was measured at 4, 24, 48 and 72 h post-electroporation (h.p.e.) and plotted as absolute values. 4 h.p.e. values are indicative of input translation and reflect transfection efficiency. Data from three independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s002.tif (12M) GUID:?36E1E99D-D384-4D9E-B100-40AE18884AB4 S3 Fig: Comparison of replication of NS5A mutants in Huh7 and Huh7.5 cells and analysis of polyprotein processing. A. WT represents the wild type mSGR-luc-JFH-1. P35A, V67A, and P145A are the mutants of domain I which can replicate at lower levels than WT in Huh7 cells; D329 is located at the C terminus of NS5A domain II. The graph shows the RLU values at 72 h.p.e. expressed as a fold increase over the 4 h.p.e. values. B. Huh7.5 cells were transfected with pCMV10-NS3-NS5B expression vectors containing the corresponding mutations. At 48 h.p.t., cell lysates were harvested in GLB and analysed by SDS-PAGE and Western blotting with anti-NS5A (sheep) and anti-NS3 (mouse). The ratio of NS5A:NS3 was calculated following quantification of Western blot signals using a Li-Cor Odyssey Sa infrared imaging system. Data from three independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s003.tif (10M) GUID:?0AF75969-47C0-41A2-97AA-696D2E953033 S4 Fig: Incucyte ZOOM visualisation of virus replication and infection. Indirect immunofluorescence analysis for NS5A expression in Huh7.5 cells electroporated with the indicated viral RNAs at 48 h.p.e. (top row). The middle row shows NS5A expression in cells infected with culture supernatants harvested from the cells presented in the top row. Infected cells were analysed at 48 h.p.i. The bottom row shows NS5A expression at 48 ZXH-3-26 h.p.i. in cells infected with cell lysates from the cells in the top rowCthis represents intracellular virus. After fixation, cells were stained with NS5A antibody and then with Alexa Fluor 568-conjugated ZXH-3-26 donkey anti-sheep IgG (red fluorescence).(TIF) ppat.1006834.s004.tif (5.5M) GUID:?589A4919-8F80-4256-9F39-8A9733A45C03 S5 Fig: Revertant and trans-complementation analysis of the phenotype of V67A and P145A in virus assembly. A. Phenotypes of V67A and P145A are not derived from acquisition of an additional compensatory mutation during the cloning process. Revertants were generated by cloning a WT NS5A fragment back into the mJFH-1 V67A or P145A mutant plasmids. Huh7.5 cells were electroporated with in vitro transcripts of the resulting V67 or P145 revertants. Virus genome replication and protein expression was assayed by quantification of NS5A positive cells 48 h.p.e. by using the Incucyte-ZOOM [62]. Intracellular and extracellular infectious virus was titrated at 72 h.p.e. B. In vitro transcribed WT JFH-1 or the indicated mutant RNAs were co-electroporated with the helper RNA (mSGR-Luc-JFH1) into Huh7.5 cells. 72 h.p.e., supernatant was harvested and cells were lysed by repetitive freeze-thaw cycles. Extracellular and intracellular virus was then titrated in Huh7.5 cells and viral infectivity was determined by using Incucyte ZOOM at 48h.p.i. Data from two independent experiments are shown and error bars represent the standard error of the mean.(TIF) ppat.1006834.s005.tif (11M) GUID:?12C502FF-D414-44EC-8329-90C92EAFA674 S6 Fig: A. Time-course immunofluorescence analysis of LD in HCV infected cells. Huh7 cells were infected with mJFH-1 WT at an M.O.I. of 0.5 ffu/cell. At the indicated h.p.e. cells had been set and stained ZXH-3-26 with BODIPY 558/568-C12, and DAPI and imaged by Airyscan microscopy. B. Colocalisation of NS5A and NS3. Quantification from the percentages of NS5A colocalized with NS3 (white blocks), or NS3 colocalised with NS5A ZXH-3-26 (crimson blocks) as proven in Fig 8. Co-localisation computations had been performed on 5 cells from at least two unbiased tests.(TIF) ppat.1006834.s006.tif (14M) GUID:?4977CFBD-4E61-41F9-8291-FA5CB598B7CF S7 Fig: Appearance of WT and domain I mutants for RNA filter binding assay. Purified cleaved domains I (35C215) analysed by SDS-PAGE and Coomassie staining (A), or Traditional western blot (B) with sheep polyclonal antiserum against NS5A.(TIF) ppat.1006834.s007.tif (3.8M) GUID:?9A597513-B131-4775-9EC0-BD20BD4E528C S8 Fig: Brief summary of the positioning and potential role of domain We mutants. Both different dimeric conformations of NS5A domains I are proven, open up (1ZH1) [15] (still left, blue/crimson) and shut (3FQM) [16], (correct, grey/crimson). P35 highlighted in aquamarine is situated in the P29CP35.