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1
Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in
cell culture
C Harak M Meyrath I Romero‐Brey C Schenk C Gondeau P Schult K Esser‐Nobis M Saeed P
Neddermann P Schnitzler D Gotthardt S Perez‐del‐Pulgar C Neumann‐Haefelin R Thimme P
Meuleman WR Vondran R De Francesco C M Rice R Bartenschlager V Lohmann
Supplementary information
Supplementary Figures 1‐11
Supplementary Table 1
Supplementary References
copy 2016 Macmillan Publishers Limited part of Springer Nature All rights reserved
SUPPLEMENTARY INFORMATIONVOLUME 2 | ARTICLE NUMBER 16247
NATURE MICROBIOLOGY | DOI 101038nmicrobiol2016247 | wwwnaturecomnaturemicrobiology 1
2
Supplementary Figure 1 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding for NS3‐5B of the indicated Con1 adaptive mutants Cells were fixed and PI4P and NS5A
were stained Fluorescent signals of whole cells were detected by confocal microscopy and
intensities of both channels of at least 30 individual cells were quantified Mean values plusmn SD (b c)
Huh7‐Lunet T7 cells with stable PI4KA knockdown (shPI4KA) or shPI4KA cells with stable HA‐PI4KA
overexpression (HA‐PI4KA) were transfected with expression constructs encoding for Con1 NS3‐5B
harboring the indicated mutations Twenty four hours after transfection cells were fixed and stained
3
for PI4P (green) and NS5A (red) Nuclei were stained with DAPI (blue b) Scale bar 10 microm PI4P
signals were quantified in at least 30 individual cells (c) Mean values plusmn SD of a representative
experiment (n=3 biological replicates) Values of NT cells for comparison were adapted from Figure
2a (d) shPI4KA or HA‐PI4KA cells were transfected with the indicated expression constructs fixed
and prepared for EM analysis 24 h after transfection (top) Diameters of at least 30 double
membrane vesicles (DMV) were determined per condition (bottom) Mean values plusmn SD Values of NT
cells for comparison were adapted from Figure 2e plt005 plt001 plt0001
4
Supplementary Figure 2 Schematic representation of different adapted replicons of gt1a1 1b2 4a3
and 6a4 Adaptive mutations corresponding to mut5A and mut5B in Con1 are shown in bold Note
that in all these replicons the mutations shown in bold were prerequisites to acquire further adaptive
mutations thus allowing the establishment of persistent replicon cultures
Supplementary Figure 3 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A NS5A was visualized by Western
Blot (see Figure 4b) and bands corresponding to basally (p56) or hyperphosphorylated (p58) NS5A
were quantified Shown is the ratio between p58 and p56 Mean values of duplicates plusmn SD (n=2) (b)
Replication data from Figure 4c and p58p56 ratios from panel a were plotted and a linear regression
analysis was performed (red line) R2 and p‐value are given Note the lack of correlation between
replication and p58p56 ratio plt005 plt001
5
Supplementary Figure 4 (a) Huh7‐Lunet T7 cells were co‐transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A and empty plasmids (‐) or plasmids
encoding for HA‐PI4KA An NS5A triple alanine mutation in the PI4KA functional interaction region
strongly impaired in PI4KA interaction was used as additional control (mutHIT5) Proteins were
metabolically labelled with [35S]‐MetCys overnight and NS5A or HA‐PI4KA was immunoprecipitated
Captured proteins were visualized by autoradiography Note that in the HA‐IP NS5A bands were
barely detectable as reported earlier5 (b) NS5A‐ and HA‐PI4KA‐specific bands from panel a were
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
2
Supplementary Figure 1 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding for NS3‐5B of the indicated Con1 adaptive mutants Cells were fixed and PI4P and NS5A
were stained Fluorescent signals of whole cells were detected by confocal microscopy and
intensities of both channels of at least 30 individual cells were quantified Mean values plusmn SD (b c)
Huh7‐Lunet T7 cells with stable PI4KA knockdown (shPI4KA) or shPI4KA cells with stable HA‐PI4KA
overexpression (HA‐PI4KA) were transfected with expression constructs encoding for Con1 NS3‐5B
harboring the indicated mutations Twenty four hours after transfection cells were fixed and stained
3
for PI4P (green) and NS5A (red) Nuclei were stained with DAPI (blue b) Scale bar 10 microm PI4P
signals were quantified in at least 30 individual cells (c) Mean values plusmn SD of a representative
experiment (n=3 biological replicates) Values of NT cells for comparison were adapted from Figure
2a (d) shPI4KA or HA‐PI4KA cells were transfected with the indicated expression constructs fixed
and prepared for EM analysis 24 h after transfection (top) Diameters of at least 30 double
membrane vesicles (DMV) were determined per condition (bottom) Mean values plusmn SD Values of NT
cells for comparison were adapted from Figure 2e plt005 plt001 plt0001
4
Supplementary Figure 2 Schematic representation of different adapted replicons of gt1a1 1b2 4a3
and 6a4 Adaptive mutations corresponding to mut5A and mut5B in Con1 are shown in bold Note
that in all these replicons the mutations shown in bold were prerequisites to acquire further adaptive
mutations thus allowing the establishment of persistent replicon cultures
Supplementary Figure 3 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A NS5A was visualized by Western
Blot (see Figure 4b) and bands corresponding to basally (p56) or hyperphosphorylated (p58) NS5A
were quantified Shown is the ratio between p58 and p56 Mean values of duplicates plusmn SD (n=2) (b)
Replication data from Figure 4c and p58p56 ratios from panel a were plotted and a linear regression
analysis was performed (red line) R2 and p‐value are given Note the lack of correlation between
replication and p58p56 ratio plt005 plt001
5
Supplementary Figure 4 (a) Huh7‐Lunet T7 cells were co‐transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A and empty plasmids (‐) or plasmids
encoding for HA‐PI4KA An NS5A triple alanine mutation in the PI4KA functional interaction region
strongly impaired in PI4KA interaction was used as additional control (mutHIT5) Proteins were
metabolically labelled with [35S]‐MetCys overnight and NS5A or HA‐PI4KA was immunoprecipitated
Captured proteins were visualized by autoradiography Note that in the HA‐IP NS5A bands were
barely detectable as reported earlier5 (b) NS5A‐ and HA‐PI4KA‐specific bands from panel a were
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
3
for PI4P (green) and NS5A (red) Nuclei were stained with DAPI (blue b) Scale bar 10 microm PI4P
signals were quantified in at least 30 individual cells (c) Mean values plusmn SD of a representative
experiment (n=3 biological replicates) Values of NT cells for comparison were adapted from Figure
2a (d) shPI4KA or HA‐PI4KA cells were transfected with the indicated expression constructs fixed
and prepared for EM analysis 24 h after transfection (top) Diameters of at least 30 double
membrane vesicles (DMV) were determined per condition (bottom) Mean values plusmn SD Values of NT
cells for comparison were adapted from Figure 2e plt005 plt001 plt0001
4
Supplementary Figure 2 Schematic representation of different adapted replicons of gt1a1 1b2 4a3
and 6a4 Adaptive mutations corresponding to mut5A and mut5B in Con1 are shown in bold Note
that in all these replicons the mutations shown in bold were prerequisites to acquire further adaptive
mutations thus allowing the establishment of persistent replicon cultures
Supplementary Figure 3 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A NS5A was visualized by Western
Blot (see Figure 4b) and bands corresponding to basally (p56) or hyperphosphorylated (p58) NS5A
were quantified Shown is the ratio between p58 and p56 Mean values of duplicates plusmn SD (n=2) (b)
Replication data from Figure 4c and p58p56 ratios from panel a were plotted and a linear regression
analysis was performed (red line) R2 and p‐value are given Note the lack of correlation between
replication and p58p56 ratio plt005 plt001
5
Supplementary Figure 4 (a) Huh7‐Lunet T7 cells were co‐transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A and empty plasmids (‐) or plasmids
encoding for HA‐PI4KA An NS5A triple alanine mutation in the PI4KA functional interaction region
strongly impaired in PI4KA interaction was used as additional control (mutHIT5) Proteins were
metabolically labelled with [35S]‐MetCys overnight and NS5A or HA‐PI4KA was immunoprecipitated
Captured proteins were visualized by autoradiography Note that in the HA‐IP NS5A bands were
barely detectable as reported earlier5 (b) NS5A‐ and HA‐PI4KA‐specific bands from panel a were
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
4
Supplementary Figure 2 Schematic representation of different adapted replicons of gt1a1 1b2 4a3
and 6a4 Adaptive mutations corresponding to mut5A and mut5B in Con1 are shown in bold Note
that in all these replicons the mutations shown in bold were prerequisites to acquire further adaptive
mutations thus allowing the establishment of persistent replicon cultures
Supplementary Figure 3 (a) Huh7‐Lunet T7 cells were transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A NS5A was visualized by Western
Blot (see Figure 4b) and bands corresponding to basally (p56) or hyperphosphorylated (p58) NS5A
were quantified Shown is the ratio between p58 and p56 Mean values of duplicates plusmn SD (n=2) (b)
Replication data from Figure 4c and p58p56 ratios from panel a were plotted and a linear regression
analysis was performed (red line) R2 and p‐value are given Note the lack of correlation between
replication and p58p56 ratio plt005 plt001
5
Supplementary Figure 4 (a) Huh7‐Lunet T7 cells were co‐transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A and empty plasmids (‐) or plasmids
encoding for HA‐PI4KA An NS5A triple alanine mutation in the PI4KA functional interaction region
strongly impaired in PI4KA interaction was used as additional control (mutHIT5) Proteins were
metabolically labelled with [35S]‐MetCys overnight and NS5A or HA‐PI4KA was immunoprecipitated
Captured proteins were visualized by autoradiography Note that in the HA‐IP NS5A bands were
barely detectable as reported earlier5 (b) NS5A‐ and HA‐PI4KA‐specific bands from panel a were
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
5
Supplementary Figure 4 (a) Huh7‐Lunet T7 cells were co‐transfected with expression constructs
encoding Con1 NS3‐5B harboring the indicated mutations in NS5A and empty plasmids (‐) or plasmids
encoding for HA‐PI4KA An NS5A triple alanine mutation in the PI4KA functional interaction region
strongly impaired in PI4KA interaction was used as additional control (mutHIT5) Proteins were
metabolically labelled with [35S]‐MetCys overnight and NS5A or HA‐PI4KA was immunoprecipitated
Captured proteins were visualized by autoradiography Note that in the HA‐IP NS5A bands were
barely detectable as reported earlier5 (b) NS5A‐ and HA‐PI4KA‐specific bands from panel a were
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
6
quantified by phospho‐imaging to calculate co‐precipitation efficiency The band of the HA‐PI4KA
input as obtained from the HA‐IP was set in relation to the band of co‐immunoprecipitated HA‐PI4KA
in the NS5A‐IP and expressed as fold wt Shown are mean values of duplicates plusmn SD (n=3) (c) Huh7‐
Lunet T7 cells were co‐transfected with expression constructs encoding for Con1 NS3‐5B harboring
the indicated mutations and with HA‐PI4KA Cells were stained for NS5A (red) and the HA‐peptide
(green) nuclei were stained with DAPI (blue) Scale bar 10 microm Whole‐cell z‐stacks were obtained by
spinning disc confocal microscopy and images were deconvolved using Huygens Essential software
and a theoretical point‐spread‐function Shown are single optical slices from deconvolved stacks (d)
Pearson correlation co‐efficients of NS5A and HA‐PI4KA from cells shown in panel c were quantified
Shown are mean values plusmn SD of at least ten different cells per condition of a representative
experiment (n=2 biological replicates)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
7
Supplementary Figure 5 (a) Huh75 cells were transfected with subgenomic Con1 wt firefly reporter
replicons and were immediately treated with the indicated inhibitor concentrations for 72 h Mean
values of triplicates plusmn SD of relative light units (RLU) measured 72 h after transfection normalized to
the DMSO control of a representative experiment (n=2 biological replicates) (b) Huh7‐Lunet cells
were mock electroporated and incubated with the indicated inhibitor concentrations for 72 h before
measurement of cellular ATP levels Mean values of triplicates plusmn SD of a representative experiment
(n=2 biological replicates) (c) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were immediately treated with the indicated inhibitor concentrations for 72 h
Note that in this experiment fixed PI4KA‐A concentrations were used and H479 was titrated in
contrast to the experiment shown in Fig 5a Mean values of triplicates plusmn SD of RLU measured 72 h
after transfection normalized to the DMSO control (n=2 biological replicates) (d) Huh7‐Lunet T7 cells
were transfected with expression constructs encoding for NS3‐5B of Con1 wt 4 h after transfection
cells were treated with the indicated inhibitor concentrations for 24 h NS5A was visualized by
Western Blot (n=1) Note that H479 reaches maximal efficiency in enhancing Con1 wt replication in
presence of PI4KA‐A at concentrations blocking p58 formation starting from 25 microM plt0001
plt001 plt005
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
8
Supplementary Figure 6 (a) Schematic representation of PI4KA inhibitors PI4KA‐B6 (purple left)
PI4KA‐F1 (green middle) and PI4KA‐G1 (orange right)7 (b d f) Huh7‐Lunet cells were transfected
with subgenomic Con1 wt firefly reporter replicons and were immediately treated with the indicated
inhibitor concentrations for 72 h Mean values of triplicates plusmn SD of RLU measured 72 h after
transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) (c e g) Huh7‐Lunet cells were mock electroporated and incubated with the indicated
inhibitor concentrations for 72 h before measurement of cellular ATP levels Mean values of
triplicates plusmn SD of a representative experiment (n=2 biological replicates) plt005 plt001
plt0001
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
9
Supplementary Figure 7 (a‐g) Huh7‐Lunet cells were transfected with subgenomic Con1 wt firefly
reporter replicons and were treated with 5 microM H479 and 1 microM PI4KA‐A (a) Treatment was started
at different timepoints after transfection Mean values of triplicates plusmn SD of relative light units (RLU)
measured 72 h after transfection of a representative experiment normalized to the DMSO control
(n=2 biological replicates) (b) Detection of Con1 wt RNA replication by Northern blot Huh7‐Lunet
cells transfected with Con1 wt replicon RNA or a replication‐deficient control (GDD) were treated
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
10
directly after transfection with the indicated inhibitors (+) or with DMSO (‐) Total RNA isolated
immediately (input) or 72 h after transfection was analyzed by Northern blot using HCV and ‐Actin
specific probes respectively The band corresponding to Con1 wt RNA is highlighted with a red
arrowhead (left panel) Band intensities of signals corresponding to Con1 wt RNA were quantified by
phospho‐imaging and normalized to input RNA and to the GDD control (right panel) Mean values plusmn
SD (n=3 2 biological and one technical replicate) (c‐e) Detection of double membrane vesicles
(DMV) in cells transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h
DMV numbers per micromsup2 (d) or DMV diameters (e) of at least five HCV‐positive cells were quantified
Mean values plusmn SD nd not detectable (f) Seventy two hours after transfection of Con1 wt replicons
and incubation with the indicated inhibitors NS5A (red) was detected by immunofluorescence
staining Nuclei were stained with DAPI (blue) Scale bar 10microm (g) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were transfected with Con1 wt replicons and treated with the indicated inhibitors for 72 h HCV
replication is indicated by nuclear GFP‐NLS arising from proteolytic cleavage of MAVS by the HCV
NS34A protease (top) The percentage of at least 50 cells with more than 60 of GFP intensity
present in the nucleus was quantified (bottom) Note that a partial nuclear localization of GFP is due
to NS34A expressed from transfected input RNA Scale bar 10microm (h) Huh7‐Lunet cells were
transfected with unadapted wt reporter constructs of the indicated genotypes and treated with the
indicated inhibitors for 72 h Mean values of triplicates plusmn SD of relative light units (RLU) measured 72
h after transfection of a representative experiment normalized to the DMSO control (n=2 biological
replicates) plt005 plt0001
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
11
Supplementary Figure 8 (a) Huh7‐Lunet cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the indicated inhibitor combination for 72 h NS5A (red)
and nuclei (blue) were stained and NS5A fluorescence intensities of at least 30 randomly chosen cells
were quantified Mean values plusmn SD of a representative experiment (n=2 biological replicates) Scale
bar 10microm (b) Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were transfected with full‐length Con1 wt RNA
genomes and were immediately treated with the combination of H479 and PI4KA‐A Twenty four
hours or 48 h after transfection compounds were selectively removed as indicated and cells were
further incubated until 72 h after transfection Intra‐ and extracellular levels of Core protein were
measured by a chemiluminescent microparticle immunoassay Shown are measurements from a
representative experiment (n=2 biological replicates) (c) Schematic of the experimental design to
assess infectivity of intra‐ or extracellular particles Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were
transfected with full‐length Con1 wt RNA genomes and were immediately treated with the indicated
inhibitors for 72 h to assess their effect on particle production As control a full‐length variant
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
12
harboring an NS5A adaptive mutation was transfected as well Seventy two hours after transfection
infectivity of cellular lysates (intracellular virus) or supernatants (extracellular virus) was assessed by
reinfecting naiumlve Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells for 2 h at 1000 g followed by additional 2 h
at 37 degC Cells were then incubated with or without the combination of H479 and PI4KA‐A for 72 h
(d) HCV RNA copies in re‐infected Huh7‐Lunet CD81 MAVS‐GFP‐NLS cells were determined by RT‐
qPCR Mean values of triplicates plusmn SD of a representative experiment normalized to the respective
DMSO controls for each condition (n=2 biological replicates) (e) Huh7‐Lunet CD81 MAVS‐GFP‐NLS
cells were infected with patient sera of two different donors (Ser1 and Ser3) tested positive for HCV
gt1b infection After infection cells were incubated with the indicated inhibitors for 72 h As control
cells were additionally treated with 100 nM Daclatasvir (DCV) Several infection events were
identified for both sera by nuclear localization of GFP‐NLS (white arrowheads) Scale bar 10microm
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
13
Supplementary Figure 9 (a) HCV RNA copies in the indicated stable Con1 replicon cell lines were
analyzed by RT‐qPCR Huh5 and Huh8 represent cell clones harboring an unmodified Con1 wt
replicon8 Huh7‐Wu mut5A cells (grey bar) were generated by transfection of Huh7‐Wu cells with
Con1 mut5A subgenomic replicons encoding for a neomycin phosphotransferase gene and
subsequent selection with G418 for 4 weeks Mean values of triplicates plusmn SD (n=2) (b) PI4KA mRNA
levels in the indicated cell lines were assessed by RT‐qPCR Mean values of triplicates plusmn SD (n=2) (c)
Huh7‐Wu Con1 wt replicon cells (Huh8) were incubated with DMSO (black bar) 001 01 025 05 1
or 5 microM of PI4KA‐A for 72 h before HCV RNA copies were quantified by RT‐qPCR Mean values of
triplicates plusmn SD normalized to the DMSO control (n=2) (d) PHH of donor 6 were infected with cell
culture‐derived (HCVcc) Con1 (gt1b) or Jc1 (gt2a) virions or with serum obtained from a chronically
infected gt1b patient Cells were then incubated with DMSO or 1 microM of PI4KA‐A for 72 h Cell viability
was assessed by total RNA yield Mean value of triplicates plusmn SD normalized to the DMSO control (e)
PHH of donor 9 and 10 were infected with patient sera tested positive for the indicated genotypes
or with cell culture‐derived JFH‐1 virus (gt2a HCVcc) and incubated with DMSO (black bar) 01 or 1
microM of PI4KA‐B for 72 h Cell viability was assessed by total RNA yield Mean value duplicates plusmn SD
normalized to the DMSO control plt005 plt0001
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
14
Supplementary Figure 10 Schematic of the proposed mode of action of PI4KA and CKIa inhibitors
(a) HCV wt in hepatoma cell lines PI4KA is activated by interaction with NS5A and NS5B while a
specific phosphorylation on NS5A possibly mediated by casein kinase I alpha (CKIa) seems to be
critical for activation and PI4P accumulation PI4KA inhibits NS5A hyperphosphorylation5 which is
also most likely mediated by CKIa9 via a so far unknown mechanism (b) Adaptive mutations in NS5A
result in a loss of the PI4KA‐activating phosphorylation pattern and of hyperphosphorylation leading
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
15
to low PI4P levels and a replication‐competent NS5A phosphorylation state (c) Inhibiting PI4KA leads
to low PI4P levels favorable for replication but on the other hand causes increased
hyperphosphorylation mediated by CKIa which is deleterious for RNA replication (d) Inhibiting CKIa
decreases both hyperphosphorylation as well as PI4KA activation possibly due to the loss of a PI4KA‐
activating phosphorylation pattern Since PI4KA is still partly active replication‐enhancing effects of
CKIa inhibition are limited (e) Combination of CKIa and PI4KA inhibition mimics the effects of
adaptive mutations in NS5A by inhibiting both PI4P accumulation and NS5A hyperphosphorylation
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
16
Supplementary Figure 11 Uncut raw images of all Western Blots and Northern Blots Subpanels are
labeled according to their appearance in main figures and supplementary figures (prefix S)
Please note that all Western Blot images were captured with a camera system therefore the raw
pictures were inverted and brightness was adjusted to visualize the bands
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
17
Supplementary Table 1 Viral loads of patient sera used in this study
Designation Figure reference Viral load (IUml)
Ser1 (gt1b) Fig 5g h Supplementary Fig 8e gt 1x108
Ser2 (gt1b) Fig 5g gt 1x108
Ser3 (gt1b) Fig 5g Supplementary Fig 8e gt 1x108
gt1b serum
(PHH donor 6) Fig 6d Supplementary Fig 9d 81x106
gt1a serum S25 Fig 6e Supplementary Fig 9e 82x106
gt1b serum S313
(PHH donor 910) Fig 6e Supplementary Fig 9e 21x107 10
gt3a serum S294
(PHH donor 10) Fig 6e Supplementary Fig 9e 23x107 10
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)
18
Supplementary References
1 BlightKJ McKeatingJA MarcotrigianoJ amp RiceCM Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture J Virol 77 3181‐3190 (2003)
2 GroblerJA et al Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase J Biol Chem 278 16741‐16746 (2003)
3 PengB et al Development of robust hepatitis C virus genotype 4 subgenomic replicons Gastroenterology 144 59‐61 (2013)
4 YuM et al Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture Antimicrob Agents Chemother 58 2638‐2646 (2014)
5 ReissS et al The lipid kinase phosphatidylinositol‐4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A PLoS Pathog 9 e1003359 (2013)
6 VaillancourtFH et al Evaluation of phosphatidylinositol‐4‐kinase IIIalpha as a hepatitis C virus drug target J Virol 86 11595‐11607 (2012)
7 BojjireddyN et al Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4‐phosphate and phosphatidylinositol 45‐bisphosphate levels J Biol Chem 289 6120‐6132 (2014)
8 BlightKJ KolykhalovAA amp RiceCM Efficient initiation of HCV RNA replication in cell culture Science 290 1972‐1974 (2000)
9 QuintavalleM SambuciniS DiPC DeFR amp NeddermannP The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation J Virol 80 11305‐11312 (2006)
10 GondeauC et al In vitro infection of primary human hepatocytes by HCV‐positive sera insights on a highly relevant model Gut 63 1490‐1500 (2014)