Metabolism of Phosphatidylinositol 4-Kinase
IIIa-Dependent PI4P Is Subverted by HCV and Is Targeted
by a 4-Anilino Quinazoline with Antiviral Activity
Annalisa Bianco1, Veronica Reghellin1, Lorena Donnici1, Simone Fenu1, Reinaldo Alvarez1,
Chiara Baruffa2, Francesco Peri2, Massimiliano Pagani1, Sergio Abrignani1, Petra Neddermann1*,
Raffaele De Francesco1*
1Department of Genomics and Molecular Biology, Virology Program, Istituto Nazionale Genetica Molecolare (INGM), Milano, Italy, 2Department of Biotechnology and
Biosciences, University of Milano Bicocca, Milano, Italy
4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was
proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline
moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by
these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase a (PI4KIIIa) has
recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-
anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIa and, to a lesser extent,
PI4KIIIb. In Huh7.5 cells, PI4KIIIa is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma
membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9,
indicating that this agent inhibits PI4KIIIa also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi
membrane, suggesting that the PI4KIIIb isoform was not significantly inhibited under our experimental conditions.
Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously
observed upon silencing PI4KIIIa by RNA interference. In light of our findings, we propose that the antiviral effect of 4-
anilino quinazoline compounds is mediated by the inhibition of PI4KIIIa and the consequent depletion of PI4P required for
the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing
significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane.
This observation implies that HCV – by recruiting PI4KIIIa in the RNA replication complex – hijacks PI4P metabolism,
ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIa product.
Citation: Bianco A, Reghellin V, Donnici L, Fenu S, Alvarez R, et al. (2012) Metabolism of Phosphatidylinositol 4-Kinase IIIa-Dependent PI4P Is Subverted by HCV
and Is Targeted by a 4-Anilino Quinazoline with Antiviral Activity. PLoS Pathog 8(3): e1002576. doi:10.1371/journal.ppat.1002576
Editor: Guangxiang George Luo, University of Kentucky College of Medicine, United States of America
Received August 16, 2011; Accepted January 26, 2012; Published March 8, 2012
Copyright: ? 2012 Bianco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work/research was partially supported by the Italian Ministry of Education, University and Research (MIUR) (grant number RBAP10TPXK to INGM).
FP acknowledges the Regione Lombardia for financial support (grant number 14546, Network Enabled Drug Design, NEDD). The funders have no role in the study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (PN); email@example.com (RDF)
Hepatitis C virus (HCV) is an enveloped, single-stranded RNA
virus classified as member of the Hepacivirus genus within the
Flaviviridae family. The 9.6 kb positive-sense RNA genome
contains a single open reading frame encoding a polyprotein of
about 3,000 amino acids, flanked by highly structured 59 and 39
untranslated (UTR) regions. Following its release into the
cytoplasm of the host cell, viral RNA is translated via an internal
ribosome entry site (IRES), giving rise to a single polypeptide that
is cleaved into 10 different mature protein products: Core, gpE1,
gpE2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. HCV
RNA replication takes place in the cytoplasm, in association with a
virus-induced intracellular membrane structure termed ‘‘membra-
nous web’’, onto which NS proteins assemble to form the so-called
RNA replication complexes.
It is estimated that 3% of the world’s population are chronically
infected by the hepatitis C virus (HCV). Most infections become
chronic and over time evolve into chronic hepatitis. The most
unwanted complication of chronic hepatitis is cirrhosis, a massive
liver fibrosis, which can lead to liver failure and hepatocellular
Since the discovery of hepatitis C virus (HCV) in the late 1980’s
much progress has been made in the understanding of the viral life
cycle of HCV. Nonetheless, to date no vaccines are available and
the current standard of care, involving lengthy treatment with a
combination of ribavirin and pegylated interferon-a (peg-IFN-a),
eradicates the infection in half of treated patients. A large effort
has been made in the past two decades in order to develop novel
anti-HCV therapies with greater efficacy. Two oral direct-acting
antiviral agents (DAA) targeting the HCV NS3/4 protease,
boceprevir and telaprevir, have recently reached the market and
PLoS Pathogens | www.plospathogens.org1 March 2012 | Volume 8 | Issue 3 | e1002576
more are being developed . While the initial efforts to the
discovery of DAA focused almost exclusively on the best
characterized HCV enzymes required for viral replication – the
NS3/4A protease and the NS5B polymerase – in the past few
years the NS5A viral protein has been attracting more and more
attention as a target for drug development [1,2]. NS5A possesses
no known enzymatic activity. It is a multifunctional non-structural
protein important for viral replication [3–6] as well as viral
assembly [7–9]. It is a phosphoprotein consisting of three domains
. Domain I is highly conserved and forms a dimeric structure
[11,12], whereas domains II and III are believed to adopt a
‘‘natively unfolded’’ conformation [13,14].
In recent years, several anti-HCV compounds identified using
cell-based replicon screens were indicated to target NS5A based
on the analysis of the mutations associated with emergence of
resistance in the replicon system [15–17].
The most studied series of these ‘‘NS5A inhibitors’’ is
represented by BMS-790052, an agent that is leading the field,
having demonstrated potent antiviral activity in clinical studies
. Compounds in this class are characterized by a complex,
dimeric or pseudo-dimeric structure and a high molecular weight,
when compared with conventional ‘‘drug-like’’ small molecules
[17,19]. Resistance mutations against these compounds emerge
readily in domain I of NS5A , with the most recurrent of these
changes corresponding to variant of tyrosine at position 93 .
Although direct interaction with purified NS5A has not been
demonstrated, compelling reverse genetic experiments  as well
as molecular models [15,21] strongly support the notion that
NS5A is the direct target of these compounds.
A less characterized series of compounds, belonging to a
different chemical class, was also initially indicated to target NS5A
on the basis of the mutation pattern observed in resistant replicons
. The common structural element of this latter class of
inhibitors is a 4-anilino quinazoline core. A representative member
of this class of compounds is A-831/AZD-2836, an experimental
antiviral agent that entered clinical trials but was later discontin-
ued due to the lack of adequate exposure . For these agents,
the mutations reported to be associated with resistance were found
to be different from those expected for the NS5A inhibitor
described above, pointing to a different mechanism of action: a
few mutations were found at the C-terminal end of NS5A domain
I (E212D, L199F and T200P), whereas most mutations occurred
in NS5A domains II and III (P299L, S370P, V388D, V362A,
S390G and S370P). Additional mutations were also found in
NS4B (S258T) and NS5B (S76A) [17,21,22]. Reverse genetics
studies in which these mutations were reintroduced in the replicon,
however, did not recapitulate the resistant phenotype observed in
the original cellular clones , leaving thus the possibility open
that these compounds act through a different viral or cellular
Interestingly, many kinase inhibitors, including some approved
antitumoral drugs (gefitinib, lapatinib, erlotinib) are 4-anilino
quinazoline derivatives [23–25]. Altogether, these considerations
led us to investigate whether the anti-HCV activity displayed by
these compounds might be due to inhibition of a cellular kinase.
Recently, several small-interfering RNA (siRNA) screening
campaigns have identified type III phosphatidylinositol 4-kinases
(PI4K) as crucial host factors for HCV replication. In particular,
PI4KIIIa was found to be required for HCV RNA replication in a
cell line- and genotype-independent manner, whereas the
requirement for the b isoform was observed to be less dramatic
and limited to Con-1 (genotype 1b) replicons [26–29]. It was
shown that the catalytic activity of PI4KIIIa is required to rescue
HCV replication in cells with a stable knock-down of PI4KIIIa. In
addition, it has been proposed that NS5A stimulates PI4KIIIa
activity by direct interaction via domain I [30–32]. All these
observations taken together made us consider the phosphatidyli-
nositol 4-kinases a potential alternative candidate target for 4-
anilino quinazoline inhibitors of HCV replication.
In this paper, we present evidence that AL-9, a member of this
class of compounds previously reported to target NS5A, inhibits
PI4P formation by direct inhibition of phosphatidylinositol 4-
kinase IIIa (PI4KIIIa). In addition, we provide evidence that
pharmacological inhibition of PI4KIIIa with AL-9 results in
altered subcellular distribution of NS5A similar to that observed
after RNAi knock-down of the PI4KIIIa mRNA, strongly
supporting a mechanism of HCV inhibition mediated by the
inhibition of PI4KIIIa. Moreover, we show that HCV subverts
components of the phosphatidylinositol-4 phosphate (PI4P)
pathway to function in favor of its own life cycle, thereby
enriching the PI4P concentration in the membranous web while
depleting the plasma membrane PI4P pool.
Compound AL-9 inhibits HCV replication in vitro
AL-9 is a member of 4-anilino quinazoline-containing HCV
replication inhibitors described previously (; Figure 1). In
order to confirm its anti-HCV activity, we tested the effect of this
compound on HCV replication in Huh7.5 cells stably expressing
genotype 1b or 2a subgenomic replicons (Con1-SR and JFH-A4,
respectively). The EC50values, calculated by measuring viral RNA
after incubation with AL-9 for three days, are reported in Table 1.
Replicon EC50values for AL-9 were found to be 0.29 mM and
0.75 mM for genotype 1b and 2a, respectively. In order to prove
that AL-9 inhibits HCV replication not only in the context of a
HCV subgenomic replicon, but also in the context of the complete
viral life-cycle, we determined the inhibitory activity using the J6/
JFH-1 HCV virus. In this case, the EC50value was found to be
1.2 mM, a figure comparable with the result obtained with
genotype 2a subgenomic replicon. CC50 values are shown for
Con1-SR, JFH-A4 and Huh7.5 cells, respectively. In summary,
It is estimated that 3% of the world’s population are
chronically infected by the hepatitis C virus (HCV). Most
infections become chronic and eventually evolve into
cirrhosis and hepatocellular carcinoma. Host factors are
interesting targets for anti-HCV therapies due to their
inherent high genetic barrier to resistance. Recently,
phosphatidylinositol 4-kinase a (PI4KIIIa) has been identi-
fied as a crucial host factor for HCV replication. Many
different pathogens, including HCV, subvert components
of the phosphatidylinositol-4 phosphate (PI4P) pathway to
function in favor of their own life cycle. In this paper, we
show that HCV dramatically alters cellular PI4P metabolism
and distribution, resulting in the enrichment of PI4P in the
membranous web required for viral replication with a
concomitant decrease of PI4P in the plasma-membrane.
Moreover, we demonstrate that 4-anilino quinazolines,
antiviral agents previously believed to target HCV NS5A,
do in fact inhibit PI4P formation by inhibition of PI4KIIIa.
This compound class is a promising lead for the
development of a novel antiviral therapy based on PI4KIIIa
inhibition. Specific PI4KIIIa inhibitors would also be
important research tools required for a deeper under-
standing of the functions and regulation of PI4P.
Anilino Quinazoline HCV Inhibitors Target PI4KIIIa
PLoS Pathogens | www.plospathogens.org2March 2012 | Volume 8 | Issue 3 | e1002576
AL-9 inhibits HCV across different genotypes with activity in the
sub-micromolar to low micromolar range in the absence of
significant cytotoxic effects.
AL-9 is an inhibitor of PI4KIIIa
In the following experiment, we investigated whether AL-9
inhibits the purified type III phosphatidylinositol 4-kinases
PI4KIIIa and PI4KIIIb (Figure 2). Both enzymes were inhibited
by AL-9 with a five-fold preference for PI4KIIIa (IC50of 0.57 mM
and 3.08 mM, respectively). This result demonstrates that AL-9
inhibits type III PI4 kinases in vitro at concentrations similar to
those required for its anti-HCV activity, displaying a moderate
selectivity for the a over the b isoform. We also tested the activity
of AL-9 on two class I PI3-kinases (p110a and p110b). While PI3-
kinase p110a was inhibited with an IC50of 1.1 mM, the potency of
AL-9 for PI3-kinase p110b was significantly lower (40% inhibition
@10 mM, data not shown).
Our hypothesis is that AL-9 inhibits HCV replication via
inhibition of PI4KIIIa. Thus, we wanted to assess whether AL-9
also inhibited PI4KIIIa in living cells. To this aim, we needed to
set up an assay that allowed us to monitor the activity of this kinase
in intact cells. PI4KIIIa is primarily localized to the ER, whereas
PI4KIIIb is localized to the Golgi membranes . It was shown
that PI4KIIIb contributes to the synthesis of PI4P at the Golgi
membranes [34,35]. Subcellular localization of the enzymes,
however, does not always coincide with their function. Thus,
PI4KIIIa, considered to be an ER-resident enzyme, has previously
been shown to be critical for the generation and maintenance of
the plasma membrane PI4P pool during phospholipase C
activation and Ca2signaling in HEK-293 or Cos-7 cells [35,36]
as well as in resting Cos-7 cells . Whether PI4KIIIa is
responsible for the maintenance of the plasma membrane PI4P
pool under normal cell culture conditions in hepatoma cells is
currently not known. Hammond et al  have developed
immunocytochemical techniques that enable selective staining of
the PI4P pool present in the plasma membrane (plasma
membrane staining protocol) or in the intracellular membranes
(Golgi staining protocol), respectively. We used this technique, in
combination with RNA gene silencing or pharmacological
inhibition, to decipher which of the type III enzymes participates
in the synthesis of the Golgi- or plasma membrane PI4P-pools in
Huh7.5 hepatoma cells.
To address which type III PI4 kinase is responsible for the
synthesis of the different cellular PI4P pools, Huh7.5 cells were
treated with siRNAs targeting PI4KIIIa, PI4KIIIb or an unrelated
siRNA (mock-siRNA) as described in the Materials and Methods
section. Immunoblots assays show specific knockdown of PI4KIIIa
or PI4KIIIb by their corresponding siRNAs (Figure 3C). Three
days after siRNA treatment, PI4P was revealed either by the
plasma membrane staining protocol (Figure 3A, upper panel) or
by the Golgi membrane staining protocol (Figure 3A, lower panel).
In cells treated with the unrelated siRNA (mock-siRNA), PI4P was
detected both in the plasma membrane and in intracellular
membranes. Intracellular PI4P was localized primarily in the
Golgi membranes, as judged by the colocalization with the Golgi
marker giantin. Silencing of PI4KIIIa resulted in a significant
decrease of the PI4P level in the plasma membrane. Concomi-
tantly with the decrease in the plasma membrane PI4P levels, we
consistently observed a pronounced increase of PI4P level in the
Golgi membrane following PI4KIIIa knockdown. In the case of
Figure 1. Chemical structure of AL-9. For the synthetic pathway
and procedure see Supporting Information.
Table 1. List of EC50values of AL-9 for different HCV
Con1-SR 1b0.29 (+/20.09)
Huh7.5+J6/JFH-1 HCV 2a
Huh7.5 cells replicating subgenomic replicons of genotype 1b or 2a (Con1-SR
and JFH-A4, respectively) or Huh7.5 cells infected with the chimeric virus J6/JFH
were treated with AL-9 for three days and intracellular viral RNA was measured
by real time PCR. The data are representative of at least three independent
experiments, and the standard deviations are shown.
*CC50measured in uninfected Huh7.5 cells.
Figure 2. Inhibitory dose-response curve of AL-9 for purified
PI4KIIIa and PI4KIIIb. The enzymes were preincubated for 10 min
with the indicated concentrations of AL-9 or DMSO and the reaction
was started by addition of 100 mM ATP and 150 mM PI:PS substrate as
described in Materials and Methods. Activity, measured as conversion of
ATP to ADP, is expressed as percent of the DMSO control. Shown is a
representative experiment of three independent experiments per-
formed in duplicate. IC50and SD of PI4KIIIa and PI4KIIIb are indicated.
Anilino Quinazoline HCV Inhibitors Target PI4KIIIa
PLoS Pathogens | www.plospathogens.org3March 2012 | Volume 8 | Issue 3 | e1002576
figure inset. Replicon ET-FAG did not replicate at appreciable
type III PI kinases. Cellular localization of PI4KIIIa (green),
PI4KIIIb (green) or NS5A (red) was analyzed by immunofluores-
cence in Huh7.5 or JFH-4A cells incubated for 4 hrs with 8 mM
AL-9 or DMSO (control). Zoomed sections are indicated by a
white square. No major effect of AL-9 on the localization of either
PI4KIIIa or PI4KIIIb was observed. Under our experimental
conditions, we observe very limited colocalization of PI4KIIIa
with NS5A (yellow) independent of the treatment with AL-9.
Effect of AL-9 on subcellular distribution of
Chemical synthesis of compound AL-9.
harboring putative resistance mutations.
Construction and assays of HCV replicons
We wish to thank Glenn Randall, Volker Lohmann and Charles M. Rice
for providing us with essential constructs and cells. We express our
gratitude to Roberto Cighetti for his contribution to the synthesis of the
compound AL-9 and to Manila Boca for her assistance and training in
operating the confocal microscope. We also would like to thank Richard
Angell and Magdalena Laver from Arrow Therapeutics for helpful
discussions during this research.
Conceived and designed the experiments: AB MP SA PN RDF. Performed
the experiments: AB RA SF VR CB LD. Analyzed the data: FP AB PN.
Wrote the paper: PN RDF.
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Anilino Quinazoline HCV Inhibitors Target PI4KIIIa
PLoS Pathogens | www.plospathogens.org17 March 2012 | Volume 8 | Issue 3 | e1002576