NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles

Fig 1. Location of mutated residues in DI. (A) Structure of NS5A illustrating the three domains. AH: amphipathic helix (blue), LCS: low complexity sequence (green). (B, C) Conserved and surface exposed residues proximal to V67 (blue) and P145 (green) are displayed in the two NS5A DI (genotype 1b) structures 1ZH1 (B) and 3FQM (C). Dimeric forms are shown on the left, with the monomers on the right.

Fig 2. Virus assembly phenotypes in Huh7.5 cells. Huh7.5 cells were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs, together with an NS5B GND mutant as negative control. Virus genome replication was analysed directly by quantification of genome copies in cell lysates using qRT-PCR, data shown have the GND values subtracted (A), and indirectly by enumerating NS5A positive cells at 72 hpe using the IncuCyte S3 (B). (C) Cell lysates were collected at 72 hpe and analysed by western blotting with the indicated antibodies. (D). Extra- and intracellular virus harvested at 72 hpe were titrated in Huh7.5 cells and quantified using the IncuCyte S3. N = 3, significant differences from WT denoted by **** (P<0.0001).

Fig 3. Co-localisation between NS5A, Core and LD. Huh7.5 cells were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs and seeded on to coverslips. At 72 hpe cells were stained with sheep anti-NS5A (white), rabbit anti-Core (green), BODIPY 558/568-C12 (red) and DAPI. Co-localisation was observed using Airyscan microscopy. Representative images are presented. A representative image of mock electroporated cells is shown in S6A Fig. Scale bars are 5 μm and 1 μm (insets).

Fig 4. Quantification of LD size and co-localisation with NS5A and Core. (A) LD numbers and size in cells from Fig 3 were calculated using Analyze Particles module of Fiji. Mock: uninfected Huh7.5 cells. 10 cells were analysed for each sample. (B) Quantification of the percentage of LD colocalized with NS5A (red), and NS5A colocalized with LD (grey). (C) Quantification of the percentage of LDs colocalized with Core (red), and Core colocalized with LD (green). (D) Quantification of the percentage of Core colocalized with NS5A (green), and NS5A colocalized with Core (grey). Co-localisation was analyzed in 10 cells for each construct using Fiji. Significant differences from WT denoted by ** (P<0.01) and **** (P<0.0001).

Fig 5. Analysis of LD distribution. (A) Cells were stained at 72 hpe with sheep anti-NS5A (white), BODIPY 558/568-C12 (red) and DAPI. (B) Distances of LDs from the nuclear membrane were evaluated using the Analyze Particles module of Fiji. 10 cells were analysed for each sample. Significant differences from WT denoted by **** (P<0.0001).

Fig 6. Virus assembly in PKR-silenced Huh7.5 cells. (A) PKR expression was detected in control Huh7.5 cells, silenced (Huh7.5 sgPKR), and cells subsequently transfected with pcDNA3.1-PKR by western blot (Huh7.5 sgPKR + PKR). (B-F) Huh7.5 cells were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs, together with an NS5B GND mutant as negative control. Virus genome replication was analysed directly by quantification of genome copies in cell lysates using qRT-PCR, data shown have the GND values subtracted (B), and indirectly by enumerating NS5A positive cells at 72 hpe using the IncuCyte S3 (C). (D) Cell lysates were collected at 72 hpe and analysed by western blotting with the indicated antibodies. (E) Extra- and intracellular virus harvested at 72 hpe were titrated in Huh7.5 cells and quantified using the IncuCyte S3. (F) Electroporated cells were treated with the PKR inhibitor C16 (1μM) from 24 hpe, extracellular virus was harvested at 72 hpe, titrated in Huh7.5 cells and quantified using the IncuCyte S3. (G) Extracellular virus harvested at 72 hpe was titrated in Huh7.5 cells and quantified using the IncuCyte S3. N = 3, significant differences from WT denoted by…

Fig 7. Co-localisation between NS5A, Core and LD in PKR-silenced Huh7.5 cells. Huh7.5 cells silenced for PKR expression were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs and seeded on to coverslips. Cells were stained at 72 hpe with sheep anti-NS5A (white), rabbit anti-Core (green), BODIPY 558/568-C12 (red) and DAPI. Co-localisation was observed using Airyscan microscopy. Representative images are shown. Representative images of mock electroporated cells are shown in S6B Fig. Scale bars are 5 μm and 1 μm (insets).

Fig 8. Quantification of LD size, abundance and co-localisation with NS5A and Core in PKR-silenced Huh7.5 cells. (A) LD numbers and size from Fig 7 were calculated using Analyze Particles module of Fiji. Mock: uninfected Huh7.5 cells. 10 cells were analysed for each sample. (B) Quantification of percentage of LDs colocalizing with NS5A (red), and NS5A colocalized with LD (grey). (C) Quantification of the percentage of LD colocalized with Core (red), and Core colocalized with LD (green). (D) Quantification of the percentage of Core colocalized with NS5A (green), and NS5A colocalized with Core (grey). Co-localisation was analyzed in 10 cells for each construct using Fiji. Significant differences from WT denoted by **** (P<0.0001).

Fig 9. Analysis of LD distribution in PKR-silenced Huh7.5 cells. (A) Cells were stained at 72 hpe with sheep anti-NS5A (white), BODIPY 558/568-C12 (red) and DAPI. (B) Distance of LDs from the nucleus was evaluated using the Analyze Particles module of Fiji. 10 cells were analysed for each sample. Significant differences from WT denoted by **** (P<0.0001).

Fig 10. Co-localisation of NS5A, dsRNA and LD. Huh7.5 cells were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs and seeded on to coverslips. At 72 hpe cells were stained with sheep anti-NS5A (green), mouse anti-dsRNA J2 (white), BODIPY 558/568-C12 (red) and DAPI. Co-localisation was observed using Airyscan microscopy. Representative images are shown. Representative images of mock electroporated cells is shown in S6C Fig. Scale bars are 5 μm and 1 μm (insets).

Fig 11. Quantification of dsRNA punctae and co-localisation with NS5A and LD. (A) Quantification of the percentage of NS5A colocalized with dsRNA (green), and dsRNA colocalized with NS5A (grey). (B) Quantification of the percentage of LD colocalized with dsRNA (red), and dsRNA colocalized with LD (grey). Co-localisation was analyzed in 10 cells from Fig 10 using Fiji. (C) Numbers of dsRNA punctae of each sample were calculated using the Analyze Particles module of Fiji. 10 cells were analysed for each sample. Significant differences from WT denoted by ns (P>0.05), **** (P<0.0001). (D) Total fluorescence of dsRNA staining was quantified by Fiji and presented as arbitrary units.

Fig 12. Co-localisation of NS5A, dsRNA and PKR. (A) Huh7.5 cells were electroporated with mJFH-1 WT and DI mutant C142A, C190A and E191A RNAs and seeded on to coverslips. At 72 hpe cells were stained with sheep anti-NS5A (red), mouse anti-dsRNA J2 (white), rabbit anti-PKR (green) and DAPI. Co-localisation was observed using Airyscan microscopy. Representative images are shown. Scale bars are 5 μm. (B) Quantification of the percentage of PKR colocalized with NS5A (green), and NS5A colocalized with PKR (red). (C) Quantification of the percentage of dsRNA colocalized with PKR (grey), and PKR colocalized with dsRNA (green). Co-localisation was analyzed in 10 cells using Fiji. Significant differences from WT denoted by **** (P<0.0001).