Recruitment and Activation of a Lipid Kinase by Hepatitis C

Transcription

Cell Host & Microbe
Article
Recruitment and Activation of a Lipid Kinase by
Hepatitis C Virus NS5A Is Essential for Integrity
of the Membranous Replication Compartment
Simon Reiss,1,13 Ilka Rebhan,1,13 Perdita Backes,1 Ines Romero-Brey,1 Holger Erfle,2 Petr Matula,3,4,10 Lars Kaderali,5
Marion Poenisch,1 Hagen Blankenburg,6 Marie-Sophie Hiet,1 Thomas Longerich,7 Sarah Diehl,6 Fidel Ramirez,6
Tamas Balla,8 Karl Rohr,3,10 Artur Kaul,1,12 Sandra Bühler,1 Rainer Pepperkok,9 Thomas Lengauer,6 Mario Albrecht,6
Roland Eils,10,11 Peter Schirmacher,7 Volker Lohmann,1,* and Ralf Bartenschlager1,*
1The
Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
RNAi Screening Facility, Heidelberg University, BioQuant, 69120 Heidelberg, Germany
3Biomedical Computer Vision Group, BioQuant, Institute of Pharmacy and Molecular Biotechnology, Department of Bioinformatics and
Functional Genomics, Heidelberg University, 69120 Heidelberg, Germany
4Center for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, CZ-60200 Brno, Czech Republic
5ViroQuant Research Group Modeling, Heidelberg University, BioQuant BQ 26, 69120 Heidelberg, Germany
6Department of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, 66123 Saarbrücken, Germany
7Department of Pathology, Heidelberg University, 69121 Heidelberg, Germany
8Section on Molecular Signal Transduction, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, MD 20892-4480, USA
9Cell Biology and Cell Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
10Department of Theoretical Bioinformatics, German Cancer Research Center, 69120 Heidelberg, Germany
11Institute of Pharmacy and Molecular Biotechnology/BioQuant, Heidelberg University, 69120 Heidelberg, Germany
12Present address: ViroLogik GmbH, 91052 Erlangen, Germany
13These authors contributed equally to this work
*Correspondence: [email protected] (V.L.), [email protected] (R.B.)
DOI 10.1016/j.chom.2010.12.002
2ViroQuant-CellNetworks
SUMMARY
Hepatitis C virus (HCV) is a major causative agent of
chronic liver disease in humans. To gain insight into
host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and
identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIa), as being
required for HCV replication. Consistent with elevated levels of the PI4KIIIa product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected
cultured hepatocytes and liver tissue from chronic
hepatitis C patients, the enzymatic activity of PI4KIIIa
was critical for HCV replication. Viral nonstructural
protein 5A (NS5A) was found to interact with PI4KIIIa
and stimulate its kinase activity. The absence of
PI4KIIIa activity induced a dramatic change in the
ultrastructural morphology of the membranous HCV
replication complex. Our analysis suggests that the
direct activation of a lipid kinase by HCV NS5A
contributes critically to the integrity of the membranous viral replication complex.
INTRODUCTION
Hepatitis C virus (HCV), the sole member of the genus Hepacivirus within the family Flaviviridae, poses a global health problem.
Treatment of infected individuals with a combination of pegy-
lated interferon and ribavirin leads to a sustained virological
response in only about 50% of patients and provokes serious
side effects. Neither a prophylactic nor a therapeutic vaccine is
available and their development has been hampered by the
high variability of the virus.
HCV has a single-stranded RNA genome of positive polarity
encoding for a polyprotein that is cleaved by cellular and viral
proteases into ten different proteins: core, envelope glycoprotein
1 (E1), E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B,
NS5A, and NS5B. The structural proteins core, E1, and E2 are
the main constituents of the virus particle, whereas most of the
nonstructural proteins are required for RNA replication (Moradpour et al., 2007; Tang and Grise, 2009). These include the
NS3/4A protein that has serine-type protease as well as NTPase
and helicase activities, the NS5B RNA-dependent RNA polymerase, and the NS5A replicase factor. The latter is composed
of three domains (Marcotrigiano and Tellinghuisen, 2009):
RNA-binding domain I, required for RNA replication; domain II,
which, to the most part, is dispensable for viral replication;
and domain III, which is essential for virion assembly (Appel
et al., 2008). NS4B is a highly hydrophobic protein triggering
rearrangements of intracellular membranes, designated the
membranous web (Egger et al., 2002). This web is composed
of membranous vesicles of heterogeneous size and morphology
and assumed to be the site of HCV RNA replication (Gosert et al.,
2003).
The possibility to propagate either subgenomic HCV replicase
constructs (replicons) or infectious HCV in cultured human hepatoma cells opened new avenues to gain insight into the intimate
interaction between HCV and its host cell (reviewed in Poenisch
and Bartenschlager, 2010). Through the use of various screening
32 Cell Host & Microbe 9, 32–45, January 20, 2011 ª2011 Elsevier Inc.
Cell Host & Microbe
PI4KIIIa Recruitment and Activation by HCV
assays, an increasing number of host factors possibly promoting
or restricting HCV replication has been described (Tai et al.,
2009; Ng et al., 2007; Supekova et al., 2008; Vaillancourt
et al., 2009; Borawski et al., 2009; Trotard et al., 2009; Randall
et al., 2007; Berger et al., 2009; Li et al., 2009; Coller et al.,
2009; Jones et al., 2010). However, the overlap of identified
genes is minimal and mechanistic insights how they contribute
to the HCV replication cycle is sparse.
Phosphoinositides (PIs) are membrane phospholipids regulating numerous cell processes by recruitment of distinct effector
proteins (D’Angelo et al., 2008). Seven PIs are known that have
been named according to the specific position in the inositol
ring where phosphorylation occurs. They are generated by
a set of kinases residing in different subcellular compartments,
thus providing a molecular signature to the membrane where
a given PI resides. A critical role of PI4kinase III (PI4KIII) for
HCV replication has been suggested earlier (Tai et al., 2009;
Vaillancourt et al., 2009; Borawski et al., 2009; Trotard et al.,
2009; Berger et al., 2009; Li et al., 2009; Hsu et al., 2010).
However, conflicting reports exist as to which specific isoform
is required, and the molecular mechanism by which PI4KIII
contributes to HCV RNA replication has not been explored in
detail.
In this study, we undertook an unbiased large-scale human
kinome screen and identified the PIP pathway as a central
element of HCV replication. We report that NS5A binds to and
activates PI4KIIIa. Local synthesis of PI4P at distinct membrane
sites appears to provide a molecular signature required for structural and functional integrity of the membranous HCV replication
complex.
RESULTS
Screening for Human Kinases Involved in HCV Entry
or Replication
To screen a small interfering RNA (siRNA) library targeting all
known and predicted 719 human kinases, we generated a suitable reporter virus system (Jc1GFP-K1402Q; described in detail
in the Supplemental Experimental Procedures available online).
Reverse transfection of siRNAs into Huh7.5 cells in a chamber
slide format was optimized according to a previously described
protocol (Erfle et al., 2007) (Supplemental Experimental Procedures). Overall, 2157 siRNAs targeting 719 human kinase genes
plus positive controls targeting the entry receptor CD81 or the
viral genome itself (HCV321 and HCV138) and four different
negative controls were spotted in transfection mixture onto
chamber slides (Figure 1A). After seeding of Huh7.5 cells, we
allowed silencing for 36 hr. Cells were then infected with
Jc1GFP-K1402Q and 36 hr later were fixed and stained with
a GFP-specific antibody. This staining was necessary because
after PFA fixation required for biosafety reasons, GFP fluorescence was too weak for reliable HCV detection, and signal to
noise ratios were too small. Fluorescence intensity was quantified by automated image analysis (Matula et al., 2009). The
screen was repeated 12 times in four independent experiments.
Data were normalized as described in the Supplemental Experimental Procedures. Figure 1B depicts the mean z scores over
the 12 replicates for each siRNA (Table S1). With a hit criterion
of 1 > z > 1 and p < 0.05, we defined 83 potential dependency
and 95 potential restriction factors for HCV entry and replication,
which were selected for further analysis.
For the validation of the 178 hit candidates, three unique
siRNAs per gene were used to minimize the number of potential
off-target hits. In addition, the format of the assay was changed
to a statistically more robust 96-well plate format to increase the
number of transfected cells per siRNA and thus statistical power
(Figure 1C). This assay format also allowed use of a renilla
luciferase (RLuc) reporter virus (JcR-ubi), facilitating the analysis
of the screen. This virus reaches infectivity titers up to 8 3
104 TCID50/ml (data not shown), and after infection of Huh7.5
cells with a MOI of 0.3 TCID50 per cell, relative light units
(RLU) were about 100-fold above background.
To determine effects on entry and RNA replication, we infected
Huh7.5 FLuc cells constitutively expressing firefly luciferase
(FLuc) with JcR-ubi 36 hr after seeding and determined replication by RLuc assay 48 hr later (Figure 1C). We normalized virusspecific RLuc counts to cell counts by determining FLuc activity
in the same lysate (see the Supplemental Experimental Procedures). The assay was repeated four times (Table S2). With
a hit criterion of 2.5 > z > 2.5 for at least two siRNAs per
gene, we identified 13 dependency factors (Figure 1D), whereas
none of the potential restriction factors could be confirmed.
To discriminate whether the identified kinases play a role in
virus entry or replication, we screened the 13 validated kinase
genes by using the HCV pseudoparticle (HCVpp) system.
z scores for each tested siRNA were calculated as described
in the Experimental Procedures (Figure 1D; summary in Table
S2). Silencing of the tested candidates did not reduce HCVpp
entry to an extent corresponding to a z score lower 2.5, arguing
for a role of these kinases in a step after virus entry such as
uncoating, establishment of the replication complex or HCV
RNA replication itself.
Bioinformatics Analysis of HCV Dependency Factors
To map those cellular pathways to which the 13 dependency
factors identified in our study are linked, we integrated various
molecular data sets, comprising information on protein-protein
interactions (PPIs), protein complexes, molecular pathways,
and functional annotations (see the Supplemental Experimental
Procedures). The inclusion of all dependency factors identified
in previous siRNA-based HCV screens enabled a meta-analysis
revealing numerous direct interactions between dependency
factors with eight of our 13 dependency factors being directly
connected to at least one previously reported dependency
factor (Table S3). Figure 2 depicts this network, and a subset
of those cellular pathways in which HCV dependency factors
are significantly overrepresented (p value < 0.05) (for detailed
information, see Table S3). These pathways include, among
others, apoptosis, integrins in angiogenesis, endocytosis, focal
adhesion, signaling in the immune system, and the ErbB
and the MAP kinase signaling pathways. The latter two have
been described by Li and colleagues to be important for HCV
and flaviviruses in general (Li et al., 2009). From our 13 dependency factors, the one having the most inhibitory siRNAs,
choline kinase alpha (CHKA), and the only factor confirmed
by all individual siRNAs, PI4KIIIa, are both associated with glycerophospholipid biosynthesis. CHKA is required for the biosynthesis of the membrane components phosphatidylcholine and
Cell Host & Microbe 9, 32–45, January 20, 2011 ª2011 Elsevier Inc. 33
Cell Host & Microbe
Figure 1. High-Throughput siRNA Screen to Identify Kinases Involved in HCV Entry and Replication
(A) Assay setup of the primary siRNA screen. Each chamber slide was coated with 384 different siRNA spots. Huh7.5 cells were infected 36 hr after seeding, and
another 36 hr later, cells were fixed and stained.
(B) Virus-specific signal intensities obtained in the primary screen were quantified and values were normalized intra- and interslide wise as well as between the 12
repetitions. Mean z scores for all siRNAs tested are depicted in gray. z scores of negative and positive controls are highlighted in blue and red, respectively. With
a hit criterion of 1 > z > 1 and a p value < 0.05 indicated by the blue and black bars, respectively, 83 potential dependency and 95 potential restriction factors
were identified (light green and light blue, respectively). Positions of PI4KIIIa hits are indicated by arrowheads.
(C) Assay setup of the entry and replication validation screen (left). For each candidate 3 unique siRNAs were tested in a 96-well plate format. Huh7.5 FLuc cells
were infected with JcR-ubi to measure effects of the silencing on HCV entry and replication (four repetitions). So that effects of the silencing of validated hits on
entry could be measured (right), Huh7.5 cells were infected with HCVpp containing a FLuc reporter gene and luciferase activity was determined 48 hr later (seven
repetitions).
(D) Summary of mean z scores of hits determined in the two independent validation screens. z scores determined in the primary screen (white bars) are shown for
comparison. The validation screens were repeated four (entry + replication) and seven (entry) times, respectively. Error bars represent the standard deviation of
the mean z -scores of the repetitions.
See also Tables S1 and S2.
Cell Host & Microbe
Figure 2. Network of HCV Dependency Factors
The depicted network highlights pathways in which dependency factors identified in our study (hexagonal nodes) and in previous HCV screens (ellipsoid nodes)
are involved. Squares represent kinases that were found in our study and have already been known as dependency factors. The mean z score of the two mostinhibitory siRNAs per gene in the entry and replication validation screen is indicated by the color intensity of the nodes. Factors in white nodes were either not
studied in this work or below the hit criterion in the primary screen; red-shaded nodes display candidates analyzed in the validation screen. Solid lines indicate
proteins that are known to interact with each other or to participate in the same protein complex. Pathways for which we found a significant enrichment of HCV
dependency factors are linked by colored areas and labeled at the top and bottom. See also Table S3.
phosphatidylethanolamine. While this result suggests that lipid
membrane biosynthesis plays a crucial role in HCV replication,
a direct interaction of CHKA with another dependency factor in
the network shown in Figure 2 is not known yet.
PI4KIIIa Is Necessary for HCV Replication
in a Genotype- and Cell Line-Independent Manner
The most consistent hit of our screen was PI4KIIIa, also known
as PIK4CA or PI4KA. In fact, it was the only gene for which all
six siRNAs tested in the primary and the validation screen scored
positive. In the initial set of experiments, we corroborated our
results from the siRNA screen and consistently found that
silencing of PI4KIIIa expression in two cell lines harboring FLuc
reporter replicons of two different HCV genotypes (JFH-1,
gt 2a and Con1ET, gt 1b) inhibited HCV RNA replication (Figures
3A and 3B). In contrast, in spite of efficient knockdown of the
beta isoform of PI4KIII, replication of the JFH-1 replicon was
not affected, and only a minor, yet statistically significant, effect
on Con1 RNA replication could be observed.
The reduction of replication measured by luciferase assay
was confirmed by immunoblotting for Con1 NS5A (Figure 3C).
The degree of silencing of PI4KIIIa expression correlated well
with the reduction of NS5A amounts, with the most effective
PI4KIIIa-specific siRNA (#3) suppressing HCV replication as efficiently as the siRNA targeting the viral genome itself (HCV321).
The same was found for JFH-1 (data not shown). Likewise,
knockdown efficiency of PI4KIIIb correlated with NS5A amounts
in Con1 replicon-containing cells (Figure 3C), whereas no effect
was found with JFH-1 replicon cells (data not shown).
We next analyzed the localization of endogenous PI4KIIIa in
cells with or without persistent subgenomic JFH-1 replicons
(neo-sgJFH-1 and mock, respectively) (Figure 3D). In both cell
pools, the kinase localized in punctuate structures throughout
the cytoplasm, but mainly in a perinuclear region, as reported
elsewhere (Kakuk et al., 2006). Staining for NS5A, a presumed
marker for HCV replication sites, revealed a striking colocalization with the kinase. In contrast, we did not observe colocalization of PI4KIIIb with NS5A (Figure 3E). In addition, also in cells
Cell Host & Microbe
Figure 3. Effect of PI4KIIIa and PI4KIIIb Silencing on HCV Replication and Localization of These Lipid Kinases
(A and B) Cells containing a genotype (gt) 2a or 1b luciferase reporter replicon specified in the top of each panel were transfected with siRNAs targeting PI4KIIIa or
PI4KIIIb (three individual siRNAs each) or with the positive control siRNA (HCV321) or the negative control siRNAs (DV-30 NTR or NegC#1). siRNAs were transfected twice, and RNA replication was determined 48 hr after the second transfection by luciferase assay. RLUs were normalized to the mean of the negative
controls. Data (mean ± SD; n = 3 in triplicates) were analyzed using a one-way t test.
(C) Lysates prepared from Con1ET replicon cells 48 hr after the second transfection were analyzed by immunoblotting for PI4KIIIa, PI4KIIIb, Transferrin receptor
(TfR), and NS5A. Levels of NS5A were quantified and normalized to the corresponding TfR levels. Values are given in percent relative to the mean of the NS5A
levels of the negative control treated cells.
(D and E) Huh7-Lunet cells (mock) or cells containing a selectable JFH-1 replicon (neo-sgJFH-1) were stained with a NS5A- (red) and either a PI4KIIIa- (D) or
a PI4KIIIb- (E) specific antibody (green). Nuclear DNA was stained with DAPI (blue). An enlargement of the sections indicated by a white square in each of the
merged images is shown in the corresponding crop panel in the bottom. Images were acquired with a confocal microscope.
with a persistent subgenomic Con1 replicon, we could observe
a colocalization of NS5A with PI4KIIIa, but not with PI4KIIIb
(data not shown).
To exclude that the dependency of HCV RNA replication on
PI4KIIIa is a cell line-specific phenomenon, we compared knockdown phenotypes in Huh7.5 and LH86 cells (Zhu et al., 2007). For
this experiment, we used a more efficient RLuc reporter virus
genome (JcR-2a) producing much higher infectivity titers as
compared to JcR-ubi and thus an about 10-fold higher signal
over background (data not shown). Silencing of PI4KIIIa clearly
decreased viral replication in both cell lines also in this infection-based assay (Figure 4A), comparable to the results obtained
with the replicon cell lines (Figures 3A and 3B). Knockdown of
PI4KIIIa expression did not affect replication of Dengue virus,
Cell Host & Microbe
arguing for a specific role of PI4KIIIa in HCV replication
(Figure 4B).
Enzymatic Activity of PI4KIIIa Is Required
for HCV Replication
For rigorous exclusion of off-target effects and to clarify whether
PI4KIIIa enzymatic activity was needed for HCV replication, we
generated Huh7.5-based cell lines with stable knockdown of
PI4KIIIa or a nontargeting control (sh-NT) (for details, see the
Supplemental Experimental Procedures). Rescue cell lines
were established by stable expression of shRNA-resistant
PI4KIIIa wild-type (WT) or the D1957A mutant, which is devoid
of enzymatic activity (Figure S1). Upon infection of the PI4KIIIawt rescue cell line with JcR-2a, HCV replication was comparable
to replication in the sh-NT control cells (Figure 4C). However, no
rescue was obtained in cells stably transduced with the inactive
PI4KIIIa D1957A mutant or an empty vector, demonstrating that
kinase activity of PI4KIIIa was required for HCV RNA replication.
PI4KIIIa Is Required for Integrity of Membranous
Web Structures
Studies on the potential contribution of PI4KIIIa to the
morphology of HCV replication sites were hampered by the
fact that interference with lipid kinase activity directly impaired
viral replication. We therefore generated stable PI4KIIIa-silenced
Huh7-Lunet cells stably expressing the T7 RNA polymerase
(Huh7-Lunet/T7), allowing the cytoplasmic expression of HCV
proteins and thus induction of membranous web formation independent of viral RNA replication. Expression of the polyprotein
fragment encoding the replicase (NS3 to NS5B) in sh-NT cells
did not affect the punctuate staining pattern of NS5A, which
was well comparable to the one found in replicon cells (compare
Figure 4D, left panel, with Figures 3D and 3E). However, silencing
of PI4KIIIa expression led to the formation of abnormal NS5A
‘‘clusters’’ (Figure 4D, right panel) in approximately 40% of the
silenced cells (Figure 4E). Importantly, this phenotype could be
rescued by overexpression of the WT kinase, but not with the
enzymatically inactive mutant. These results suggested that
enzymatic activity of PI4KIIIa was necessary for integrity of the
membranous replication complex.
Ultrastructural analyses of the same sh-NT cells expressing the
NS3 to NS5B polyprotein revealed membrane rearrangements
with accumulation of heterogeneous vesicles (Figure 4F) resembling the membranous web detected in Jc1-infected cells (data
not shown). Prominent membrane rearrangements only detected
in cells expressing the HCV polyprotein (compare to Figure S2)
included double-membrane vesicles (DMVs) with an average
diameter of 211 ± 71 nm and multimembrane vesicles (MMVs)
with an average diameter of 287 ± 85 nm. In contrast, NS3 to
NS5B expression in sh-PI4KIIIa cells did not trigger formation
of MMVs. In addition, the DMVs were smaller in size, very homogeneous (134 ± 30 nm) and aggregated into huge clusters resembling the phenotype observed in immunofluorescence analysis.
Elevated PI4P Levels in HCV-Containing Cells In Vitro
and In Vivo
As reported previously, PI4P, the product synthesized by
PI4kinases, was detected predominantly in the Golgi membrane
(Figure 5A) (colocalization with the Golgi marker TGN46 not
shown) (reviewed in D’Angelo et al., 2008). Upon infection with
HCV, a profound and time-dependent altered subcellular distribution as well as an increase of PI4P was found resulting in
a pattern that very much resembled HCV replication sites. In
fact, a fraction of NS5A-positive dot like structures (19.7% ±
4.4% SEM) colocalized with PI4P, arguing that the kinase generated this product at HCV replication sites. In contrast, Huh7 cells
harboring a DENV replicon neither showed this relocalization of
PI4P nor a colocalization of PI4P with assumed sites of viral replication (data not shown).
Quantitative analysis of PI4P immunofluorescence in HCVinfected cells revealed an 3-fold induction in PI4P levels
compared to mock-treated cells (Figure 5B). Consistently, we
found that PI4P levels in neo-sgCon1-, neo-sgJFH-, and Jc1transfected cells were increased 3-fold compared to mock
cells when measuring PI4P levels in an immunoblot assay (Figure 5B). Importantly, PI4KIIIa protein levels were not elevated
by HCV RNA transfection or infection (Figure 5C), arguing for
activation of PI4KIIIa by HCV (Figure 5C).
We furthermore investigated PI4P levels in hepatocytes of
patients persistently infected with HCV by using immunohistochemistry on consecutive sections of frozen liver samples (Figure 5D and Figure S3; Tables S4 and S5). NS5A- or core-reactive
cells were only found in infected tissues obtained from HCVpositive patients, demonstrating specificity of the staining
method. Importantly, staining for NS5A, core, and PI4P revealed
that HCV-positive areas were always enriched in PI4P, thus
corroborating our in vitro data and indicating that HCV infection
indeed resulted in elevated PI4P levels.
Role of NS5A Domain 1 in Recruitment of PI4KIIIa
and Induction of PI4P
Given the colocalization of PI4KIIIa with NS5A and the accumulation of PI4P at sites of HCV replication, we assumed that the
kinase might be recruited by interaction with one or several
replicase proteins. Individual HCV proteins were therefore
coexpressed with HA-tagged PI4KIIIa in Huh7-Lunet/T7 cells,
and coimmunoprecipitation experiments were performed with
monospecific antisera (Figure 6A). We found that NS5A and
NS5B, but not the NS3/4A protease/helicase or NS4B, interacted
with the kinase. Moreover, expression of NS5A, but not NS5B,
lead to an induction and altered staining pattern of PI4P (Figure S4), and this pattern was well comparable to the one observed
in infected cells (Figure 5A). These results suggested that NS5A is
the key player altering PI4P amounts and subcellular distribution,
most likely by modulating PI4KIIIa activity.
To identify the region of NS5A interacting with the kinase, we
performed pulldown experiments by using NS3 to NS5B polyproteins containing different in frame deletions in NS5A (Figure 6B) (Appel et al., 2008; Backes et al., 2010). Only by deleting
domain I did we observe a reduced interaction between
NS5A and PI4KIIIa. In addition, the deletion of NS5A domain I
also hampered the induction of PI4P, whereas deletions of
domain II or III had no effect (Figure 6C). We thus concluded
that NS5A domain I is required for interaction with PI4KIIIa.
Activation of PI4KIIIa Kinase Activity by NS5A
We next investigated whether HCV proteins directly affected
PI4KIIIa activity by using in vitro kinase assays and
Cell Host & Microbe
Figure 4. Impact of PI4KIIIa Silencing on HCV Production and Integrity of the Membranous Web
(A) Expression of PI4KIIIa in Huh7.5 or LH86 cells was silenced with individual siRNAs, and cells were infected with JcR-2a. siRNA HCV321 served as positive
control, and siRNAs DV-30 NTR and NegC#1 were used as negative controls. Virus replication was determined by luciferase assay. Data (mean ± SD; n = 2 in
duplicates) were analyzed with a one-way t test.
(B) Same as in (A) but measuring Dengue virus replication with a Renilla reporter virus that will be described elsewhere. Data (mean ± SD; n = 2 in duplicates) were
analyzed with a one-way t test. p values below 0.05 or 0.001 are indicated by one or three asterisks, respectively.
(C) Huh7.5 cells with stable knockdown of PI4KIIIa expression (sh-PI4KIIIa) or expressing a nontargeting shRNA (sh-NT) were stably transduced with shRNAresistant PI4KIIIa wild-type (wt) or D1957A mutant (inactive) expression constructs. For comparison, the PI4KIIIa knockdown cell line was transduced with
the empty expression vector in parallel (empty vector). Cell lines were additionally transfected with siRNA #3 targeting PI4KIIIa (corresponding to the sequence
of the shRNA used for stable silencing) prior to infection with JcR-2A with a MOI of 0.5 TCID50/cell. Virus replication was determined by luciferase assay 48 hr after
infection. As negative control, sh-NT cells were infected and analyzed in parallel. Mean values and standard deviations of triplicate values from one representative
experiment of four independent experiments are shown.
Cell Host & Microbe
recombinant-purified NS3, NS5A, or NS5B (Figure 7A). Incubation of the kinase with a phosphatidylinositol-containing
substrate and radiolabeled g-[32P] ATP led to clearly detectable
incorporation of radioactive phosphate into phosphatidylinositol
(Figure 7B). Incorporation of radioactivity could be reduced by
addition of Wortmannin (WM), a known inhibitor of the kinase
(Balla et al., 1997), to levels obtained with an inactive mutant (Figure S1), thus confirming specificity of the assay. Addition of NS3
or NS5B only slightly reduced or increased kinase activity,
respectively (Figure 7B). In contrast, NS5A enhanced PI4KIIIa
activity dose dependently up to 5-fold. This stimulation was
abolished by Wortmannin, excluding an irrelevant kinase activity
copurified with NS5A.
Finally, we analyzed the effect of the PI4KIIIa inhibitor PIK93 on
the subcellular distribution of PI4P in HCV-containing cells.
PIK93 treatment inhibited the HCV-mediated PI4P induction
and, in addition, caused a clustering phenotype of NS5A very
much like the clustering observed upon knockdown of PI4KIIIa
expression (Figure 7C).
Since PIK93 is not specific for PI4KIIIa and inhibits primarily
the beta isoform (Borawski et al., 2009), we analyzed the
distribution of PI4P also by specific silencing of the alpha
isoform. Importantly, we found no colocalization of PI4P with
membrane clusters induced by silencing of PI4KIIIa (Figure 7C).
PI4P levels were increased 6-fold in naive cells expressing
the HCV replicase proteins but remained unaltered upon
silencing of PI4KIIIa expression (data not shown). These results
confirmed that kinase activity was required for elevated PI4P
levels.
Taken together, the data suggest that PI4KIIIa is recruited to
the membranous replication compartment by NS5A, which in
turn stimulates kinase activity. Elevated PI4P amounts generated at these sites appear to directly contribute to membranous
web integrity.
DISCUSSION
Focusing on kinases that often play key roles in regulating viral
replication, in this study, we used an RNA interference (RNAi)based screen targeting the human kinome and identified 13
kinases promoting HCV RNA replication. Up to now, nine limited
and two genome-wide siRNA screens based on different HCV
replication models have been performed (Tai et al., 2009;
Ng et al., 2007; Supekova et al., 2008; Vaillancourt et al.,
2009; Borawski et al., 2009; Trotard et al., 2009; Randall et al.,
2007; Berger et al., 2009; Li et al., 2009; Coller et al., 2009; Jones
et al., 2010). However, the overlap of identified genes is very low,
which may be due to different experimental conditions, reagents,
or hit calling criteria. Nevertheless, bioinformatic analysis of our
results in the context of published HCV dependency factors
provides a comprehensive overview about kinase-linked pathways involved in HCV entry and replication (Figure 2). Eight of
our 13 hits were each directly connected to at least one previously reported dependency factor, arguing for a high quality of
our screen. Most notably, a comparison of host cell factors
involved in HCV, DENV, and West Nile virus replication revealed
three commonly involved pathways (MAPK signaling pathway,
focal adhesion, and ErbB signaling pathway), demonstrating
the close evolutionary relationship of these three viruses (Li
et al., 2009; Krishnan et al., 2008).
Kinases reported earlier to be involved in HCV replication and
identified also in our screen include casein kinases I and II
(Neddermann et al., 2004; Kim et al., 1999; Tellinghuisen et al.,
2008; Masaki et al., 2008) and choline kinase alpha (CHKA)
(Li et al., 2009). The latter is required for the biosynthesis of phosphatidylethanolamine and phosphatidylcholine, a major component of lipid membranes, arguing that this enzyme contributes to
the formation of the membranous web. However, no change in
the localization pattern of CHKA after HCV infection, and no
colocalization of this kinase with HCV replication sites was
found, arguing that the kinase may contribute to replication
more indirectly (data not shown).
The most consistent hit identified in our screen was PI4KIIIa.
This enzyme is one of four kinases (PI4KIIa, PI4KIIb, PI4KIIIa,
and PI4KIIIb) in mammalian cells that catalyze the synthesis of
PI4P (Balla and Balla, 2006). All four enzymes have different
subcellular localizations and regulation mechanisms of their
activity state, thus producing distinct PI4P pools inside the cell
(reviewed in D’Angelo et al., 2008). PI4KIIIa has also been
identified by others as HCV dependency factor (Tai et al.,
2009; Vaillancourt et al., 2009; Borawski et al., 2009; Trotard
et al., 2009; Berger et al., 2009; Li et al., 2009). In agreement
with these studies, we found that silencing of PI4KIIIa inhibits
HCV RNA replication. Conflicting results have been obtained
for the beta isoform of this lipid kinase. We found that PI4KIIIb
promotes replication of HCV genotype 1b (Con1), but not of
a genotype 2a (JFH-1) genome, arguing for genotype-specific
dependency.
PI4KIIIa appears to localize primarily at the endoplasmic reticulum (ER) (Kakuk et al., 2006; Wong et al., 1997), assumed to be
the origin of HCV replication sites. Indeed, we observed a clear
colocalization of endogenous PI4KIIIa with NS5A, although the
subcellular distribution of the kinase was not dramatically
changed in HCV-containing cells (Figure 3D). PI4KIIIb is primarily
(D) Huh7-Lunet/T7 cells stably expressing a PI4KIIIa-specific shRNA (sh-PI4KIIIa) or a nontargeting shRNA (sh-NT) (as described in C) were transfected with a T7
promoter-based NS3 to NS5B expression construct. Cells were stained with a NS5A-specific antibody (red) and nuclear DNA was stained with DAPI (blue). Note
the formation of profound NS5A ‘‘clusters’’ in PI4KIIIa knockdown cells, but not in sh-NT cells.
(E) Cell lines described in (C) were transfected with a NS3 to NS5B polyprotein expression construct and the percentage of ‘‘wildtype’’ and ‘‘cluster’’ phenotype
(D) was determined for 250 HCV-positive cells per condition.
(F) Control (top) and PI4KIIIa-silenced cells (bottom) were transfected with a NS3 to NS5B expression construct containing a functional NS5A with a GFP insertion
in domain III (Schaller et al., 2007) and prepared for EM analysis. The GFP insertion had no impact on the morphology of membrane alterations induced by the
HCV NS proteins in IF or EM (not shown) and does not interfere with RNA replication in a replicon context (Schaller et al., 2007). Consecutive enlargements of the
boxed areas are shown from left to right. Note the very heterogeneous membranous web (MW) in control cells and the clusters of small double-membrane vesicles (DMVs) (indicated by yellow arrows) in sh-PI4KIIIa cells. Scale bars are given in the lower right of each panel. N, nucleus; LD, lipid droplet; MMVs, multimembrane vesicles; rER, rough endoplasmic reticulum; m, mitochondrium.
See also Figures S1 and S2.
Cell Host & Microbe
Figure 5. PI4P and PI4KIIIa Levels in HCV-Containing Cells
(A) HCV-permissive Lunet/CD81 cells were mock treated or infected with Jc1. Cells were fixed at given time points and stained with a NS5A- (red) and a PI4Pspecific antibody (green). Nuclear DNA was stained with DAPI (blue). An enlargement of the section indicated by the white square is shown in the crop panel in the
bottom. Images were acquired with a confocal microscope.
(B) Quantification of PI4P levels by immunofluorescence analysis (green bars) and immunoblot (black bars). For immunofluorescence of mock- or Jc1-transfected
cells, mean values and SEM of 80 cells per condition are shown. For immunoblot, cells transfected with Jc1 or subgenomic replicons specified in the bottom were
lysed after 48 hr. Lipids were extracted and spotted onto membranes, and PI4P was quantified with the PI4P Mass Strip Kit (Echelon). Signals were quantified with
the Quantity One software (Bio-Rad). Error bars represent the SEM of three independent experiments. IF, immunofluorescence; IB, immunoblotting; n.t., not
tested.
(C) Lysates used in (B) were prepared from mock- or Jc1-transfected cells at time points post transfection given in the top and analyzed by immunoblotting for
PI4KIIIa, NS5A, and Calnexin (loading control).
(D) Immunohistochemistry on snap-frozen liver tissues from a HCV-negative person (10H6; Tables S4 and S5) or a patient with chronic HCV infection (3E6; Tables
S4 and S5). Consecutive liver sections were stained for NS5A, PI4P, and core. Note that cells staining positive for viral proteins and PI4P reside in the same region
of each section.
See also Figures S3 and S4 and Tables S4 and S5.
localized to the Golgi compartment (Figure 3E) (De Matteis et al.,
2005), hampering kinase recruitment by HCV proteins and
explaining the lack of colocalization with viral proteins (Figure 3E).
The different subcellular localization of both isoforms might also
explain why silencing of the expression of the alpha isoform
cannot be rescued by expression of the beta isoform (Tai
et al., 2009). However, since our results suggest that PI4P might
have a central role in mediating the function of PI4 kinases in
Cell Host & Microbe
Figure 6. Role of NS5A Domain I in Recruitment of PI4KIIIa and Induction of PI4P
(A) Huh7-Lunet/T7 cells were cotransfected with HA-tagged PI4KIIIa and expression constructs encoding individual viral proteins specified in the top. After 8 hr,
cell proteins were radiolabeled for 16 hr with [35S] methionine/cysteine-containing medium. Cells were lysed, and immunoprecipitation was performed with the
antibodies indicated in the bottom. Filled arrowheads point to HCV proteins; the open arrowhead marks PI4KIIIa.
(B) Huh7-Lunet/T7 cells were cotransfected with HA-tagged PI4KIIIa and pTM-based NS3 to NS5B polyprotein constructs containing given deletions of individual
NS5A domains. Immunoprecipitation was performed as described in (A) with an NS5A-specific antibody. Filled arrowheads on the right refer to NS5A variants; the
open arrowhead marks PI4KIIIa.
(C) Huh7-Lunet/T7 cells were transfected with constructs encoding NS3 to NS5B harboring wild-type NS5A or NS5A deletion mutants specified in the top. Twenty
four hours after transfection, NS5A (red) and PI4P (green) were detected by immunofluorescence. Nuclear DNA was stained with DAPI (blue). Images were
acquired with a confocal microscope. Numbers in the bottom refer to percentage and SEM of NS5A-positive dot-like structures costaining with PI4P. Values
are derived from analysis of 20 individual cells.
See also Figure S4.
HCV replication, subtle differences in the subcellular localization
and dynamics of PI4P pools in different cell lines may account for
the controversial knockdown phenotypes observed for the PI4K
isoforms.
Results obtained by immunohistochemical staining of serial
sections of frozen liver tissues from patients with chronic hepatitis C were in full support of our in vitro data. This technique
that allowed us to detect the viral antigens core and NS5A is
superior to the use of paraffin-embedded tissues, where detection of HCV antigens frequently fails (Shiha et al., 2005). We
observed varying proportions of cells (up to a maximum of about
5%) staining positive for HCV in different patient samples (Table
S4). Liang and colleagues, who used two-photon microscopy in
combination with virus-specific, fluorescent, semiconductor
Cell Host & Microbe
Figure 7. Activation of PI4KIIIa by NS5A
(A) Analysis of recombinantly expressed and
purified HCV nonstructural proteins and PI4KIIIa.
NS3 and NS5A were expressed as full-length
proteins with N-terminal hexahistidine tag, whereas
NS5B was C-terminally tagged and lacked the
C-terminal membrane insertion sequence. One
microgram of each protein was loaded onto
a SDS 7% polyacrylamide gel, and proteins were
visualized by Coomassie blue staining.
(B) Purified PI4KIIIa was incubated with a phosphatidylinositol-containing substrate and radiolabeled g-[32P] ATP in the presence or absence of
the indicated purified viral proteins. Molar ratios
of PI4KIIIa and a given viral protein are indicated
in the bottom. PI4KIIIa activity was determined
by measurement of the incorporation of [32P] into
the phosphatidylinositol substrate. As controls,
the PI4KIIIa inhibitor Wortmannin (WM; 100 mM)
was added to the reaction. Data (mean ± SD;
n = 3 in duplicates) were analyzed with a twoway t test. p values below 0.05 or 0.001 are indicated by one or three asterisks, respectively.
n.d., not detectable.
(C) Naive Huh7-Lunet/T7 cells or cells with stable
knockdown of PI4KIIIa expression (sh-PI4KIIIa) or
expressing a nontargeting shRNA (sh-NT) were
transfected with a NS3 to NS5B polyprotein
expression construct under control of the T7
RNA polymerase promoter (pTM-NS3-5B) and
analyzed for NS5A (red) and PI4P (green) distribution. A fraction of the cells were treated with 30 mM
PIK93 for 16 hr. Mock-transfected Huh7-Lunet/T7
cells (left column of panels) are shown for reference. Nuclear DNA was stained with DAPI (blue).
Images were acquired with a confocal microscope. White arrows point to a regular Golgi-like
distribution of PI4P.
See also Figure S1.
quantum dot probes, recently reported that 7% to 20% of hepatocytes in infected liver tissue are HCV positive (Liang et al.,
2009). The lower frequency of HCV antigen-positive cells identified by our method might point to lower sensitivity, thus detecting only cells with high antigen load. Nevertheless, we found that
HCV-positive regions in the infected livers were always enriched
in PI4P, thus supporting our in vitro data.
So far, we do not know whether HCV replication depends on
PI4KIIIa itself or on its reaction product, PI4P. On one hand,
PI4P might be a direct constituent of the membranes forming
the HCV replication complex; on the other hand, PI4P might
specifically recruit host cell proteins required for HCV RNA replication. One potential candidate is the oxysterol binding protein,
which binds to PI4P and is essential for HCV RNA replication
(Amako et al., 2009). We note that a similar model has recently
been proposed for generation of the replication compartment
of enteroviruses (Hsu et al., 2010). Upon infection with coxsackie
virus, secretory pathway organelles are reorganized leading to
the formation of a PI4P lipid-enriched microenvironment in
a PI4KIIIb-dependent manner. Interestingly, the viral RNAdependent RNA polymerase of coxsackie virus specifically binds
to PI4P, supporting the notion that PI4P serves as recruitment
factor required for the assembly of the membrane-associated
replication complex. In agreement with our observation, Hsu
and coworkers also reported a slight increase of PI4P in HCV
replicon cells and colocalization of PI4P with NS5A. However,
in contrast to what we found, these authors described that
knockdown of PI4KIIIb reduced HCV RNA replication much
stronger as compared to reducing PI4KIIIa expression. This
discrepancy might be due, at least in part, to the use of only
a genotype 1b replicon for their experiments.
Our knowledge on the biogenesis of the membranous web is
still incremental. In principle, NS4B is capable of inducing
membrane alterations reminiscent of the membranous web
(Egger et al., 2002; Gosert et al., 2003) and might provide the
protein scaffold of these vesicles. However, more recent results
(Ferraris et al., 2010) and the data presented in our study point to
a far more complex biogenesis that includes the formation of
heterogeneous DMVs and MMVs. Although NS5A does not
trigger vesicle formation by itself (Egger et al., 2002), our data
point to an essential role of NS5A for membranous web integrity
by activating PI4KIIIa, leading to an accumulation of PI4P at sites
of HCV replication. Whether the altered lipid composition
induced in this way contributes to membranous web formation
Cell Host & Microbe
or whether this is due to viral or host proteins binding to these
lipids remains speculative and will be the subject of more
detailed studies.
By using stable knockdown of PI4KIIIa expression and reconstitution experiments, we demonstrate that kinase activity of
PI4KIIIa is required for efficient HCV RNA replication. Although
earlier studies performed with the kinase inhibitors Wortmannin
and PIK93 lead to the same conclusion, the pleiotropic effects
imposed by these drugs and their cytotoxicity preclude rigorous
confirmation of this assumption. Both inhibitors are not exclusively targeting PI4KIIIa (Tai et al., 2009; Borawski et al., 2009).
Importantly, we demonstrate elevated levels of PI4P in HCVcontaining cells both in cell culture and in vivo and provide
convincing evidence that NS5A binds to and activates PI4KIIIa
activity. On the basis of these results, we propose a model in
which infecting RNA genomes are translated at the rough ER
(rER), giving rise to high amounts of polyprotein (Quinkert
et al., 2005). NS5A generated by polyprotein cleavage binds to
the kinase in a domain I-dependent manner and recruits the
enzyme to ER-derived membranes. Binding of NS5A to the
kinase stimulates its activity, resulting in high levels of PI4P at
these membrane sites. Those membranes thus obtain a PIP
signature, which might contribute directly to membrane properties or recruit viral or host factors required for proper architecture
and functionality of the membranous web.
In conclusion, we have identified kinases and pathways
involved in HCV replication, most notably the PIP pathway. Our
data provide a molecular mechanism by which PI4KIIIa contributes to HCV RNA replication and how elevated PI4P amounts
are induced by viral infection. Given the crucial role of PI4
kinases in HCV replication, they represent possible targets for
development of antiviral therapy. Moreover, the perturbation of
the PIP pathway induced by HCV infection may impact cell-cycle
control and thus contribute to the formation of hepatocellular
carcinoma.
EXPERIMENTAL PROCEDURES
Cell Lines, Viruses, and Plasmid Constructs
For infection experiments the highly permissive cell lines Huh7.5 (Blight et al.,
2002), LH86 (Zhu et al., 2007), Lunet/CD81 (Koutsoudakis et al., 2007), or
Huh7.5 FLuc was used. The latter was derived from Huh7.5 cells by stable
retroviral transduction of the gene encoding for the Firefly luciferase (FLuc)
via a transduction approach described elsewhere (Koutsoudakis et al.,
2007). PTM vectors allowing expression of the HCV nonstructural proteins
NS3 to 5B with or without deletions of NS5A subdomains as well as individual
HCV NS proteins have been described recently (Backes et al., 2010). All
sequences were derived from the JFH-1 isolate. The gene encoding the
230 kDa full-length PI4KIIIa isoform 2 was obtained from Kazusa DNA
Research Institute, Chiba, Japan (product ID FXC00322, corresponding to
GenBank accession number AB384703). Further details about cell lines and
constructs are given in the Supplemental Experimental Procedures.
siRNA Screening Protocols
The primary screen targeted 719 human kinases with three siRNAs each and
by reverse transfection on chambered coverglass slides (Lab-Tek chamberslides, Thermo-Scientific) with an image-based readout. The validation screen
(entry and replication) targeted 178 hit candidates with three siRNAs each and
by reverse transfection in a 96-well plate format. The HCVpp-based validation
screen targeted 13 hit candidates with three siRNAs each and by reverse
transfection in a 96-well plate format. Detailed information can be found in
the Supplemental Experimental Procedures.
PI4P Quantification
Huh7-Lunet cells were electroporated with 10 mg of the respective viral RNA
transcript and seeded into 6-well plates. After 48 hr, cells were lysed, and
PI4P was extracted with the PI4P Mass Strip Kit (Echelon) according to the
manufacturer’s instructions. Extracted PI4P was spotted onto membranes
and detected with the PI4P Mass Strip Kit and the ECL-plus reagent
(Amersham). Quantification of PI4P amounts was carried out with the Quantity
One software package (Bio-Rad). PI4P staining observed by immunofluorescence was quantified with the Image J program. A z projection of confocal
z stacks was generated with the ‘‘sum slices’’ option. After thresholding of
the signal intensity of PI4P staining, PI4P amount was determined by defining
cell areas and taking the IntDen-value obtained with the ‘‘analyze particles’’
functions of Image J.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on serial sections obtained from
snap-frozen liver tissue using the DAKO EnVison System HRP (DAKO, K4004).
Cryosections were fixed for 20 min at RT with prewarmed 4% PFA and washed
two times in PBS. Blocking and antigen detection were performed according
to the manufacturer’s instructions with the following modifications: PBS was
used for all washing steps, primary antibodies were incubated for 1 hr at
room temperature (RT), and secondary antibodies were incubated for 45 min
at RT. NS5A mouse monoclonal antibody 9E10 (generous gift of C.M. Rice)
was used at a final concentration of 0.5 mg/ml, core monoclonal antibody
C7-50 (Moradpour et al., 1996) at 2 mg/ml, and the PI4P-specific IgM monoclonal antibody (Echelon, Z-P004) at 1 mg/ml. Sections were counterstained
with hematoxylin and mounted with Aqua-Tex (Merck). Human tissue samples
were provided by the Tissue bank of the National Center for Tumor Diseases
Heidelberg after approval by the ethics committee.
Phosphatidylinositol-Kinase Assay
The lipid kinase assay measured the incorporation of phosphate into a phosphatidylinositol-containing substrate. Fifty-microliter reactions were supplied
with 1 mCi g-[32P] ATP (Perkin Elmer), 10 mM nonradioactive ATP, 10 mM
PI:PS (Invitrogen; PV5122), 13 kinase buffer T (20 mM Tris/HCl [pH7.4],
5 mM MgCl2, 0.5 mM EGTA, and 0.4% Triton X-100), 2 mM DTT, 5 ml
DMSO, and 1 mg PI4KIIIa (generous gift of K. Lin and B. Wiedmann) (Borawski
et al., 2009). As a control, the PI4KIIIa inhibitor Wortmannin (100 mM; SigmaAldrich; W1628) was added. Increasing amounts of NS5A, NS3, or NS5B
were added to the reactions. After 45 min at RT, the assay was stopped by
addition of 200 ml 1 M HCl, and lipids were extracted with 400 ml of a 1:1 chloroform:methanol suspension. After rigorous mixing and centrifugation
(13,000 rpm for 2 min in a table-top centrifuge), 125 ml of the organic phase
was transferred to a scintillation vial and mixed with 4 ml UltimaGold (Perkin
Elmer). Incorporation of radioactive phosphate was measured by liquid scintillation counting.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and five tables and can be found with this article online at
doi:10.1016/j.chom.2010.12.002.
ACKNOWLEDGMENTS
We are grateful to Ulrike Herian, Stephanie Kallis, Rahel Klein, Jürgen Beneke,
and Nina Beil for excellent technical assistance. We thank Wolfgang Fischl for
providing the DENV reporter construct and Charles M. Rice for the Huh7.5 cells
and the NS5A monoclonal antibody 9E10. We are grateful to Kai Lin and
Brigitte Wiedmann for providing purified PI4KIIIa and PIK93. We thank Jeremy
Luban and Thomas Pertel for stable knockdown vector and Mark Harris for the
NS5A polyclonal sheep serum. We thank Rossella Pellegrino for her assistance
with IHC. We are grateful to the Nikon Imaging Center at the University of
Heidelberg for providing access to their facility. This work was supported in
part by the research program RNS/RNAi of the Landesstiftung BadenWürttemberg (P-LS-RNS/30 to R.B.), a grant from the FORSYS ViroQuant
project (BMBF, FKZ 0313923 to R.B. and V.L.), and the German Research
Foundation DFG (SFB/TRR77, Teilprojekt A1 to R.B. and V.L., Teilprojekt B5
Cell Host & Microbe
to T.L. and P.S.; grant Lo 1556/1-1 to V.L and KFO 129/1-2 to T.L. and M.A.).
The ViroQuant-CellNetworks RNAi Screening core facility was supported by
CellNetworks—Cluster of Excellence (EXC81). The work directed by M.A.
was supported by the German National Genome Research Network NGFN
(BMBF, contract number 01GR0453) and the DFG-funded Cluster of Excellence for Multimodal Computing and Interaction. The work of P.M. was funded
by the FORSYS ViroQuant project and The Ministry of Education of the Czech
Republic, grant numbers 2B06052 and MSM0021622419. M.P. is supported
by an intramural grant of the Medical Faculty of the University of Heidelberg,
and M.-S.H. is supported by a Marie-Curie fellowship from the EU (EI-HCV).
The work related to liver biopsy analysis was supported by the Tissue bank
of the National Center for Tumor Diseases Heidelberg. R.B. and members of
the Molecular Virology unit in Heidelberg would like to dedicate this study to
the memory of Professor Heinz Schaller, a wonderful mentor and colleague
and a generous sponsor, who made this unit and the work conducted therein
possible and who shaped Heidelberg Life Sciences so much.
Received: September 24, 2010
Revised: November 12, 2010
Accepted: December 16, 2010
Published: January 19, 2011
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Recruitment and Activation of a Lipid Kinase by Hepatitis C

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