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Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCVParticle ProductionRoesch, Kathrin; Kwiatkowski, Marcel; Hofmann, Sarah; Schoebel, Anja; Gruettner, Cordula;Wurlitzer, Marcus; Schlueter, Hartmut; Herker, EvaPublished in:Cell reports
DOI:10.1016/j.celrep.2016.08.052
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Citation for published version (APA):Roesch, K., Kwiatkowski, M., Hofmann, S., Schoebel, A., Gruettner, C., Wurlitzer, M., ... Herker, E. (2016).Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV ParticleProduction. Cell reports, 16(12), 3219-3231. https://doi.org/10.1016/j.celrep.2016.08.052
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Article
Quantitative Lipid Droplet
Proteome AnalysisIdentifies Annexin A3 as a Cofactor for HCV ParticleProductionGraphical Abstract
Highlights
d Quantitative lipid droplet proteome analysis of HCV-infected
cells is presented
d ANXA3 is recruited to lipid-rich fractions in HCV-infected cells
d ANXA3 functions as a host factor required for efficient HCV
particle production
d ANXA3 promotes virion maturation by facilitating
incorporation of apolipoprotein E
Rosch et al., 2016, Cell Reports 16, 3219–3231September 20, 2016 ª 2016 The Author(s).http://dx.doi.org/10.1016/j.celrep.2016.08.052
Authors
Kathrin Rosch, Marcel Kwiatkowski,
Sarah Hofmann, ..., Marcus Wurlitzer,
Hartmut Schl€uter, Eva Herker
In Brief
Cytoplasmic lipid droplets are vital to
hepatitis C virus particle production.
Rosch et al. report a quantitative lipid
droplet proteome analysis and identify
annexin A3 as a regulator of HCV particle
maturation and egress.
Accession Numbers
PXD004707
Cell Reports
Article
Quantitative Lipid Droplet Proteome AnalysisIdentifies Annexin A3 as a Cofactorfor HCV Particle ProductionKathrin Rosch,1 Marcel Kwiatkowski,2 Sarah Hofmann,1 Anja Schobel,1 Cordula Gr€uttner,1 Marcus Wurlitzer,2
Hartmut Schl€uter,2 and Eva Herker1,3,*1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany2Core Facility Mass Spectrometric Proteomics, Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, 20246
Hamburg, Germany3Lead Contact*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2016.08.052
SUMMARY
Lipid droplets are vital to hepatitis C virus (HCV)infection as the putative sites of virion assembly,but morphogenesis and egress of virions remain illdefined. We performed quantitative lipid dropletproteome analysis of HCV-infected cells to identifyco-factors of that process. Our results demonstratethat HCV disconnects lipid droplets from their meta-bolic function. Annexin A3 (ANXA3), a protein en-riched in lipid droplet fractions, strongly impactedHCV replication and was characterized further:ANXA3 is recruited to lipid-rich fractions in HCV-in-fected cells by the viral core and NS5A proteins.ANXA3 knockdown does not affect HCVRNA replica-tion but severely impairs virion production with lowerspecific infectivity and higher density of secretedvirions. ANXA3 is essential for the interaction of viralenvelope E2 with apolipoprotein E (ApoE) and fortrafficking, but not lipidation, of ApoE in HCV-in-fected cells. Thus, we identified ANXA3 as a regulatorof HCV maturation and egress.
INTRODUCTION
Hepatitis C virus (HCV) infection is one of the leading causes of
liver-related morbidity and mortality worldwide, accounting for
approximately 0.5 million deaths every year (Wedemeyer et al.,
2015). The true number of HCV infections is unknown, but recent
estimates suggest 80 (64–103) million viraemic HCV infections,
and the disease burden will likely continue to rise in most coun-
tries (Gower et al., 2014). No vaccine exists, but the recently
developed direct-acting antivirals dramatically increase thera-
peutic responses compared to the standard interferon-based
therapy. However, worldwide, the treatment of patients will
likely be restricted due to the extremely high costs of the new
therapeutics.
Cell ReportThis is an open access article under the CC BY-N
HCV belongs to the family of Flaviviridae. The enveloped viral
particles contain a single positive-stranded RNA genome of
9.6 kb in length and are associated with lipoproteins and neutral
lipids and accordingly named lipoviroparticles. After receptor-
mediated endocytosis, fusion, and uncoating, the viral genome
is translated into one polyprotein precursor (reviewed in Linden-
bach and Rice, 2005). Host and viral proteases process the viral
polyprotein, releasing the three structural proteins (nucleocapsid
core, E1, and E2), the viroporin p7, and six non-structural (NS)
proteins (NS2, 3, 4A, 4B, 5A, and 5B). Multi-protein RNA replica-
tion complexes containing minimally NS3–5B proteins replicate
the viral RNA within ER (endoplasmic reticulum)-derived struc-
tures termed the membranous web. The membranous web
contains single-, double-, and multi-membrane vesicles (Ro-
mero-Brey et al., 2012), as well as cytosolic lipid droplets that
may serve as viral assembly sites (reviewed in Lindenbach and
Rice, 2013). After encapsidation of newly synthesized viral
RNA at ER membranes near lipid droplets, the virus is thought
to exit the cell via the secretory pathway, thereby maturing
to low-density lipoviroparticles (reviewed in Lindenbach and
Rice, 2013).
In the absence of full viral replication, only two viral proteins
localize to lipid droplets, core and NS5A (Barba et al., 1997;
Shi et al., 2002). All other viral proteins are found in close prox-
imity to lipid droplets in infected cells (Miyanari et al., 2007),
but they lack intrinsic lipid-droplet-targeting features, as they
fail to localize to lipid droplets when expressed individually (Ca-
mus et al., 2013). Translocation of both core and NS5A to lipid
droplets requires triglyceride biosynthesis as inhibitors of diacyl-
glycerol acyltransferase-1 (DGAT1) impair trafficking to lipid
droplets and subsequent HCV assembly (Camus et al., 2013;
Herker et al., 2010). Proper processing of core is a prerequisite
for core’s ability to traffic to lipid droplets (Targett-Adams
et al., 2008), and mutations in either core or NS5A that disrupt
lipid droplet binding suppress HCV assembly (Boulant et al.,
2007; Miyanari et al., 2007). Trafficking of core to lipid droplets
additionally requires cytosolic phospholipase A2 activity (Menzel
et al., 2012), and lipid-droplet-binding proteins act as host
factors with, e.g., PLIN3/TIP47 required for both HCV RNA repli-
cation and release of virions (Ploen et al., 2013a, 2013b; Vogt
s 16, 3219–3231, September 20, 2016 ª 2016 The Author(s). 3219C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A B
Lysis Lipid Droplets 3x
WashSDS-PAGE
LiDLDLLD ss
NS4A A5SN3SNCore mKO2 BSD NS5BNS4BE1 E2 NS2p7
Jc1 NS5AB-mKO2-BSD NS5ABCleavage Sites
Heavy AA Light AA
6 passages
6–10 days+/- blasticidin
selection MS
Tubulin
PLIN2/ADRP
Calreticulin
Lysate LDs
PLIN3/TIP47
MnSOD
C LDsM
D E
G
6Enrichment at lipid droplets(log2 HCV-infected/control)
0 2 4-2-4-6
DDX3XC14orf166DDX1IGF2BP1RTCBPABPC1PAPSS2DPP4ANXA3SERPINH1ARL8BVIMCTSDVDAC2MGST1MTTPGGHATP1A1SSR1SFPQVDAC1VAT1P4HBMYDGFERP29ARF4
***************
*
*****
EPHX1DDX5ATL3RAB32FAM213ARPS13DDOSTRPN2CAPRIN1ATP5A1PCYOX1RPN1TMED10ATP5BPHB2LMAN2CANXVDAC3TPRG1LRHOCSRSF1ETFAHM13RPS11RPS4XCALR
METTL7A ***ACACA **ACSL4 *PNPLA3 **APOBVPS13AFASN *LSS *ABHD5 **LMNB1 **NUS1PNPLA2 *HSD17B7 *ACSL3 *PSAT1DHRS1 ***CHP1 ***STMN1 *FABP5 *IPO5PRKDC *GBF1SCCPDH *HSPB1LDHA **SQLE *LDAHELMOD2AHCYNSDHLDHRSX *HSP90AB1 *PHGDH
tRNA splicing, via endonucleolytic cleavage and ligation
endoplasmic reticulum lumencytoplasmic stress granule
cytoplasmic part
tRNA-splicing ligase complexmembrane-bounded vesicle
RNA bindingchylomicron-mediated lipid transport
negative regulation of sequestering of triglyceridetriglyceride metabolic process
long-chain fatty-acyl-CoA biosynthetic processcholesterol biosynthetic process
endoplasmic reticulumlipid particle
oxidoreductase activitysteroid biosynthesis
fatty acid biosynthesisfatty acid metabolism
metabolic pathwaysPPAR signaling pathway
ChREBP activates metabolic gene expressionfatty Acyl-CoA biosynthesis
acyl chain remodeling of DAG and TAGmetabolism of lipids and lipoproteins
BPBP
CCCCMF
KEGGREACREACREACREAC
KEGGKEGGKEGGKEGG
BPBP
BP
CCCCMFREAC
CC
CCCC
-log10 p-value0 2 4
PAPSS2
C14orf166
DDX1
IGF2BP1
RTCBPABPC1
DPP4
ANXA3
SERPINH1ARL8B
VIM
MTTP
P4HB
ERP29
ARF4
HSP90AB1
DHRSX
SQLE
LDHA
SCCPDH
PRKDC FABP5STMN1
CHP1
DHRS1
ACSL3
HSD17B7
PNPLA2LMNB1 ABHD5
LSS
FASN
PNPLA3ACSL4
ACACA
METTL7A
F
shDDX1shDPP4shRAB32shHMGCS1shLMAN2shPABPC1shVIL1shANXA3shARF4
shNTnaive
HCV replication(shTarget/shNT)
0 0.5 1 1.5
Figure 1. Quantitative Lipid Droplet Prote-
ome Analysis of HCV-Infected versus Unin-
fected Control Cells
(A) Huh7.5 cells cultured in media containing either
heavy or light amino acids were infected with an
HCV reporter virus. Lipid droplets were isolated by
two sequential sucrose gradient centrifugations,
washed, and subjected to LC-ESI-MS/MS.
(B) Western blot of the lipid droplet (LD) fraction
shows enrichment of the lipid-droplet-binding pro-
teins PLIN2/ADRP and PLIN3/TIP47. Other subcel-
lular compartment markers as calreticulin (ER),
MnSOD (mitochondria), or b-tubulin were unde-
tectable in lipid droplet fractions.
(C) Coomassie-stained SDS-PAGE of a purified lipid
droplet (LD) fraction (M, molecular weight marker).
(D) Heatmap depicting enriched or depleted
proteins (1.5-fold cutoff). *p < 0.05; **p < 0.01;
***p < 0.001.
(E) Gene ontology (GO) enrichment analysis of
annotations for molecular function (MF), cellular
compartment (CC), and biological process (BP), as
well as biological pathways through KEGG and
Reactome (REAC) annotations. Shown are annota-
tions significantly enriched and hierarchically
filtered for enriched (red) and depleted (blue) lipid
droplet proteins. Analysis was performed using the
gProfileR package in R.
(F) Protein interaction networks of dysregulated
lipid-droplet-associated proteins. Shown is the
interaction network of significantly enriched (red) or
depleted (blue) proteins visualized using the cisPath
package in R.
(G) Huh7.5 cells were transduced with lentiviral
stocks expressing the indicated shRNAs, followed
by infection with HCV Jc1NS5AB-EGFP. 6 days post-
infection, cells were fixed and analyzed by flow
cytometry of EGFP to measure HCV infection rates.
Shown is the relative infection normalized to the
non-targeting shRNA (shNT) control (mean ± SD,
n = 2). Red indicates proteins that are enriched at
lipid droplets.
See also Figure S1 and Tables S1, S2, S3, S4, S5,
S6, and S7.
et al., 2013). Despite recent advances, the mechanistic details of
the late stages of HCV replication are still ill defined.
Here, we performed an extensive quantitative lipid-droplet
proteome analysis of HCV-infected cells to identify host factors
for HCV particle production. One of the highly enriched proteins
was annexin A3 (ANXA3), a member of the annexin family of cal-
cium-dependent, phospholipid-binding proteins. All annexins
show cytosolic andmembrane localizations, and some including
ANXA3 are also secreted. They are involved in endo- and exocy-
tosis and trafficking, and they serve as membrane scaffolds
organizing specific membranemicrodomains (reviewed in Gerke
et al., 2005). Membrane recruitment is most likely regulated by
calcium influx, and, depending on the phospholipid-binding
specificity, annexins may target different cellular membranes
(reviewed in Gerke et al., 2005; Gerke and Moss, 2002). We
found that ANXA3 is specifically recruited to lipid-rich fractions
in cells infected with HCV and that it participates in HCVmatura-
tion and release. Thus, by performing quantitative lipid droplet
3220 Cell Reports 16, 3219–3231, September 20, 2016
proteomics, we revealed that HCV disconnects lipid droplets
from their metabolic function and identified ANXA3 as a host
factor for HCV replication.
RESULTS
HCV Infection Profoundly Changes the Lipid DropletProteomeTo identify regulators of HCV replication, we performed quanti-
tative lipid droplet proteome analysis of HCV-infected versus
uninfected control cells, using stable isotope labeling by amino
acids in cell culture (SILAC) (Figure 1A). We labeled HCV-
permissive Huh7.5 cells with heavy amino acids for at least
six passages until near-complete (>95%) incorporation of the
heavy amino acids into the cellular proteins was achieved. Cells
cultured in media containing the normal light amino acids were
infected with HCV carrying a fluorescence reporter (mono-
meric Kusabira Orange 2; mKO2) to monitor infection rates,
followed by a blasticidin resistance gene (blasticidin S deami-
nase [BSD]) in between a duplicated NS5A-NS5B cleavage
site described previously (Webster et al., 2013). After 2 to
3 weeks, HCV-infected cells that displayed infection rates
higher than 95% were used for the experiments. Of note, we
used selected HCV-infected cells in half of the experiments.
One day prior to lipid droplet isolation, HCV-infected and
heavy-amino-acid-labeled control cells were plated at equal
cell densities. For lipid droplet isolation, 5 3 107 cells were
mixed and lysed in hypotonic lysis buffer with a Dounce
homogenizer. Lipid droplets were isolated by two sequential
sucrose density centrifugations of post-nuclear supernatant,
followed by three washing steps in isotonic buffer. The isolated
lipid droplet fractions were subsequently analyzed by western
blotting to confirm enrichment of lipid-droplet-binding proteins
(PLIN2/ADRP and PLIN3/TIP47) (Bickel et al., 2009) and de-
pletion of markers of other cellular compartments (Figure 1B).
SDS-PAGE followed by Coomassie staining was used to sepa-
rate the lipid droplet fractions (Figure 1C), and after tryptic
digest, peptides were analyzed by liquid chromatography-elec-
trospray ionization-tandem mass spectrometry (LC-ESI-MS/
MS). As a control, we switched labeling conditions in one
experiment, thus culturing the HCV-infected cells with heavy
amino acids to exclude isotope-specific effects.
In four independent experiments (on two different MS plat-
forms), we identified around 1,500 proteins, of which 316 were
detected through multiple peptides in each experiment and
selected for further analysis (datasets are listed in Tables S1,
S2, S3, and S4).
To correct for different cell numbers or numbers of lipid drop-
lets, we next centered the detection ratios of light over heavy
peptides (L/H) or vice versa in swapped labeling conditions
(H/L) by dividing through the median of the identified proteins
(Table S5). Correlation analysis over all identified proteins
revealed reproducible protein quantification independent of the
labeling conditions, blasticidin selection, or MS platform used,
with a mean Pearson correlation coefficient of r = 0.54 for all ex-
periments (Figure S1). Detection ratios of the identified proteins
were then ranked according to their mean enrichment at lipid
droplets in the four independent experiments. For further anal-
ysis, we used cutoff ratios of greater than 1.5-fold enrichment
or depletion and selected the proteins with significantly altered
abundance (Figure S1). This generated a list of 16 proteins that
are enriched significantly in lipid droplet fractions of HCV-in-
fected cells and 21 proteins that are depleted; these proteins
showed similar ratios of lipid droplet localization in all four inde-
pendent experiments (Figure 1D).
Next, we conducted gene ontology (GO) enrichment analysis
and further characterized the lipid droplet proteome of HCV
infection (Figure 1E; Tables S6 and S7). Recruited proteins are
enriched for proteins usually not associated with lipid droplet
function, such as RNA-binding proteins and proteins associated
with cytoplasmic stress granules and vesicles. Intriguingly, the
depleted proteins are enriched for lipid metabolic annotations
such as lipid particle as the cellular compartment and fatty
acid and triglyceride metabolism for biological processes;
thus, proteins generally associated with lipid droplets are
depleted from lipid droplets in HCV-infected cells. Protein
network analysis highlights the protein clusters identified both
for enriched and for depleted proteins (Figure 1F).
ANXA3 Is Required for Efficient HCVSpreading InfectionTo validate our approach of identifying regulators of HCV replica-
tion, we used short hairpin RNA (shRNA)-mediated knockdown
of a subset of proteins detected at lipid droplets, some of which
were enriched in HCV-infected cells. We transduced Huh7.5
cells with lentiviral particles encoding the shRNAs and simulta-
neously expressing mCherry to monitor transduction efficacy.
Subsequently, cells were infected with a fluorescently labeled
HCV reporter virus (Jc1NS5AB-EGFP) to identify proteins that
impact HCV replication. Among them, knockdown of ANXA3
and ARF4 (ADP-ribosylation factor 4) showed the strongest
effect on HCV replication (Figure 1G). As ARF4 is a brefeldin A
(BFA)-sensitive ARF, and previous studies had investigated the
effect of BFA on HCV replication, we focused on ANXA3 in the
present study. In infection experiments, Jc1NS5AB-EGFP efficiently
spread in mock- and control shRNA-transduced cells, while
knockdown of ANXA3 severely impaired viral spreading (Figures
2A–2D). Knockdown of ANXA3 was confirmed by qRT-PCR and
western blotting (Figures 2B and 2C). Previous studies had iden-
tified a different annexin, ANXA2, as a host factor for HCV RNA
replication or virion assembly (Backes et al., 2010; Saxena
et al., 2012), while others only found a minor effect (Dreux
et al., 2012). We detected ANXA2, -3, -4, and -5 in lipid droplet
fractions, but only ANXA3 was enriched in HCV-infected cells
(Table S5). We wanted to clarify the impact of knocking down
each of the ANXA proteins on viral replication and found that,
in our experimental setup, only ANXA3 knockdown efficiently
blocked spreading of Jc1NS5AB-EGFP reporter viruses (Figures
2E and 2F).
To independently confirm a role for ANXA3 in HCV replication,
we created clonal knockout cell lines using the CRISPR (clus-
tered regularly interspaced short palindromic repeats)/Cas9 sys-
tem (Figure 2G). Huh7.5 cells were transiently transfected with a
Cas9 expression plasmid encoding single guide RNA (sgRNA)
targeting the ANXA3 locus right after the translation start codon.
After single-cell cloning, indels were verified by sequencing (Fig-
ure S2), and ANXA3 knockout was confirmed bywestern blotting
(Figure 2H). We generated three cell lines harboring ANXA3
deletions and one cell line that transiently expressed the Cas9
construct but still harbored the wild-type alleles. When we in-
fected these cell lines with Jc1NS5AB-mKO2 reporter viruses,
spreading infection was nearly completely blocked in the
ANXA3-knockout cell lines as compared to the wild-type control
clone or the parental Huh7.5 cells (Figure 2I). We also analyzed
this phenotype by fluorescence microscopy, and, indeed,
Jc1NS5AB-mKO2 was impaired from spreading in DANXA3 cells,
as indicated by the very low number of infected cells per foci
of infection as compared to the control cells (Figure 2J).
ANXA3 Is Recruited to Lipid-Rich Fractions inHCV-Infected CellsIn order to confirm the re-localization of ANXA3 to lipid droplet
fractions in HCV infection, we isolated lipid droplets of naive
and HCV-infected Huh7.5 cells by sucrose gradient centrifuga-
tion and analyzed ANXA3 levels via western blotting. Despite
Cell Reports 16, 3219–3231, September 20, 2016 3221
A B
D
NS4A NS5BEGFPNS5ANS3Core NS4BE1 E2 NS2p7
Jc1 NS5AB-EGFPshRNA(mCherry)
Flow cytometryHuh7.5
0
100
20
40
60
80
EG
FP-p
ositi
ve c
ells
(%)
day 2 day 4 day 6
*shNTMock
shANXA3
shN
T
Moc
k
ANXA3
shA
NX
A3
Tubulin
*
C
AN
XA
3ex
pres
sion
0 0.2 0.4 0.6 0.8 1.0 1.2
shNTshANXA3
day 3 day 5
G
Huh7.5
Cas9sgRNA
NS4A NS5BmKO2NS5ANS3Core NS4BE1 E2 NS2p7
Jc1 NS5AB-mKO2
0
25
5
10
15
20
mK
O2-
posi
tive
cells
(%)
day 2 day 4 day 6
**WTParental
ΔANXA3
*
***
**
Cel
l num
ber /
foci
0
10
20
30
40
50
Parental ΔANXA3WT
** ****
Par
enta
lΔ
AN
XA
3W
T
Tubulin
ANXA3
Par
enta
l CRISPR Clones
*
ΔANXA3 WT
#1 #2 #3 #4
H
I J
E F
AN
XA
exp
ress
ion
0 0.2 0.4 0.6 0.8 1.0 1.2
AN
XA
2 A
NX
A3
AN
XA
4 A
NX
A5
EG
FP-p
ositi
ve c
ells
(%)
shANXA2 shANXA3 shANXA4 shANXA5
shNTMock
day 2 day 4 day 6 0
100
20
40
60
80
Figure 2. ANXA3 Controls HCV Replication(A) Scheme of the experimental design using ANXA3-knockdown cells.
(B) Knockdown efficacy was verified by qRT-PCR.
(C) Western blot analysis of shANXA3 (shRNA targeting ANXA3)-transduced and control cells. Asterisk indicates an unspecific band recognized by the ANXA3
antibody.
(D) Huh7.5 cells were transduced with lentiviral stocks expressing shRNAs targeting ANXA3 (shANXA3) or non-targeting (NT) control (shNT) and infected
with Jc1NS5AB-EGFP. 2, 4, and 6 days post-infection, cells were fixed and analyzed by flow cytometry of EGFP tomeasure spreading infection (mean ± SEM, n = 3).
*p < 0.05.
(E) HCV spreading infection in ANXA2–5 knockdown cells. Shown is one experiment performed in triplicate (mean ± SD).
(F) Knockdown efficacies were verified by qRT-PCR.
(G) Scheme of the experimental design using ANXA3-knockout cells.
(H) Western blot of ANXA3-knockout and wild-type clones as well as parental Huh7.5 cell lines. Asterisk marks an unspecific band.
(I) ANXA3-knockout (clone #3), wild-type (clone #4), and parental Huh7.5 cells were infected with Jc1NS5AB-mKO2 viral stocks and analyzed by flow cytometry of
mKO2 to measure spreading infection (mean ± SEM, n = 3). *p < 0.05; **p < 0.01.
(J) Confluent ANXA3-knockout (clone #3), wild-type (clone #4), and parental Huh7.5 cells were infected with a very low MOI of Jc1NS5AB-mKO2 and fixed 3 days
post-infection for microscopic analysis. Cells were stained with NS5A antibodies and counterstained with Hoechst (scale bar, 10 mm). The number of infected
cells per infection focus was counted in two independent experiments in two to three different wells (mean ± SEM). **p < 0.01.
See also Figure S2.
3222 Cell Reports 16, 3219–3231, September 20, 2016
A
B
C D
E F
Figure 3. ANXA4 Translocates to Lipid
Droplet Fractions in HCV-Infected Cells
(A) Huh7.5 cells were infected with
Jc1NS5AB-EGFP-BSD and selected for infected cells
with blasticidin. Lipid droplets (LDs) were isolated
by sucrose gradient centrifugation and analyzed
by western blotting.
(B) Western blot analysis of ANXA3 expression in
Huh7.5 cells during HCV infection.
(C) Huh7.5 cells were transfected with Con1
subgenomic replicon (Con1 SGR) RNA and kept
under selective pressure with G418. Lipid drop-
lets were isolated by sucrose gradient centrifu-
gation and analyzed by western blotting.
(D) Membrane flotation assays using iodixanol
gradients of replicon-transfected and control
cells. Fractions were collected top (1) to bottom
(8) and analyzed by western blotting. The asterisk
marks an unspecific band.
(E) Lipid droplet fractions of Huh7.5 cells trans-
duced with lentiviral expression constructs for
FLAG-core or NS5A-FLAG were analyzed by
western blotting (arrow indicates ANXA3, asterisk
marks an unspecific band).
(F)Huh7.5cellswereelectroporatedwithexpression
constructs for ANXA3-HA and NS5A-FLAG or
FLAG-core, fixed, and stained with HA and FLAG
antibodies, followed by lipid droplet staining with
LD540. Samples were analyzed by confocal micro-
scopy;singlechannelsareshowninblackandwhite,
themerged images are pseudocolored as indicated
(scale bars, 10 mm). Co-localization of the viral pro-
teins (VP) core and NS5A with ANXA3-HA was
analyzed using coloc2 in Fiji (Schindelin et al., 2012).
We analyzed individual cells of three independent
experiments and calculated the degree of co-local-
ization using Manders’ co-localization coefficient
andPearson’s correlation coefficient (mean± SEM).
loading the same amount of protein as indicated by similar
PLIN2/ADRP levels, ANXA3 was nearly undetectable in lipid
droplet fractions from control cells but readily detectable in lipid
droplet fractions from HCV-infected cells (Figure 3A). Therefore,
ANXA3 is localized to lipid droplet fractions in cells that are in-
fected with HCV. This increase in ANXA3 levels could either
reflect an overall increased expression or a specific recruitment
of ANXA3 to lipid droplets. Therefore, we analyzed ANXA3
expression following HCV infection by western blotting. Overall,
ANXA3 protein expression was unchanged in infected cells, as
compared to control cells, pointing to enhanced recruitment of
Cell Report
ANXA3 to lipid-rich fractions following
infection with HCV (Figure 3B).
Next, we investigated ANXA3 localiza-
tion in cells transfected with a bicistronic
Con1 subgenomic replicon (Con1 SGR)
that lacks all structural proteins, including
core, as well as p7 and NS2, and only
expresses NS3 to NS5B, as well as a
neomycin resistance gene (Choi et al.,
2004). After selection for cells that replicate
HCV RNA, we isolated lipid droplets of
replicon and control cells and analyzed thembywestern blotting.
As observed before, ANXA3 was barely detectable in the lipid
droplet fraction of control cells (Figure 3C). In contrast, in cells
selected for expression of the subgenomic HCV replicon and ex-
pressing the NS3-5B RNA replicase, ANXA3 was recruited to the
lipid droplet fraction (Figure 3C). The HCV RNA is replicated
within the membranous web. PLIN3/TIP47 is a lipid-droplet-
binding protein that acts as a host factor for HCVRNA replication
and is re-localized to low-density lipid-rich membranes in
membrane floatation assays (Vogt et al., 2013). Hence, we
analyzed ANXA3 localization in HCV RNA—replicating cells by
s 16, 3219–3231, September 20, 2016 3223
membrane floatation assay. Post-nuclear cellular lysates were
separated by a discontinuous iodixanol gradient and analyzed
by western blotting. ANXA3 localized to high-density mem-
branes but additionally co-fractionated with NS5A and PLIN2/
ADRP in low-density lipid-rich membranes in cells actively
replicating HCV RNA (Figure 3D). Thus, expression of NS3-
NS5B and active viral RNA replication are sufficient to re-localize
ANXA3 to lipid-rich membranes.
To analyze which viral protein re-localizes ANXA3, we
ectopically expressed the two viral proteins that traffic to lipid
droplets independently of viral replication, core and NS5A,
and performed lipid droplet isolations. In control cells,
ANXA3 was barely detectable in lipid droplet fractions. In
contrast, ANXA3 traffics to lipid-rich fractions in cells express-
ing either core or NS5A (Figure 3E). We next assessed co-
localization of both proteins together with hemagglutinin
(HA)-tagged ANXA3 in immunofluorescence microscopy. We
could not analyze endogenous ANXA3, as all antibodies
tested failed to specifically detect endogenous ANXA3 in
immunofluorescence staining. Overexpressed ANXA3-HA
mainly showed cytoplasmic staining pattern. Co-localization
analysis through quantifying co-localization according to
Manders and Pearson revealed that ANXA3-HA partially co-
localized both with NS5A and with core protein (Figure 3F)
but did not display clear ring-like lipid droplet staining
patterns. Taken together, the data indicate that ANXA3 is spe-
cifically recruited to lipid-rich membrane fractions by core and
NS5A during HCV infection.
HCV RNA Replication Is Independent of ANXA3Next, we dissected which step of viral replication depends on
ANXA3 expression. As ANXA3 is recruited to lipid-rich mem-
brane fractions in cells replicating a subgenomic replicon RNA,
we first used the Con1 SGR system to analyze viral RNA replica-
tion in cells harboring ANXA3 shRNA (Figure 4A). Con1 SGRRNA
was transfected into Huh7.5 cells transduced with the different
shRNAs. After 3 weeks in selection media, growing cell colonies
were visualized by crystal violet staining. We observed slightly
fewer colonies in ANXA3-knockdown cells as compared to con-
trol cells (Figure 4A). In order to verify this result, we used a
monocistronic genotype 2a replicon system containing a firefly
luciferase in between a duplicated NS5A-NS5B cleavage site
and partially deleted in E1 and E2 to prevent viral spreading
(Jc1DE1E2NS5AB-Fluc; Figure 4B) (Webster et al., 2013). In
contrast to the subgenomic replicon, this system is very sensitive
and allows the examination of viral RNA replication in short-term
experiments. In-vitro-transcribed Jc1DE1E2NS5AB-Fluc RNA was
transfected into ANXA3-knockdown or control cells and
subjected to luciferase assays at different time points post-elec-
troporation. To normalize to the transfection efficacy, all values
are expressed as fold increase of luciferase activity per micro-
gram of protein over a 4-hr time point before viral RNA replication
occurs. Compared to the control, we could not detect any differ-
ences in viral RNA replication rates in ANXA3-knockdown cells
(Figure 4B). In addition, we analyzed the viral protein expression
in ANXA3-knockdown and control cells after electroporation of
wild-type JFH1 RNA by western blotting. ANXA3-knockdown
and control cells showed comparable levels of core and NS5A,
3224 Cell Reports 16, 3219–3231, September 20, 2016
indicating that viral RNA replication and translation are not
affected by knockdown of ANXA3 (Figure 4C).
ANXA3 Is Required for Efficient HCV Particle ProductionWe next asked whether ANXA3 influences the later stages of
HCV replication. We transfected full-length HCV Jc1NS5AB-EGFP
genomes into ANXA3-knockdown or control cells and confirmed
equal transfection efficacy by flow cytometry of EGFP. We then
isolated total cellular RNA and viral RNA from the culture super-
natant and measured HCV copy numbers by qRT-PCR at day 3
and day 6 post-transfection. In line with the previous experi-
ments, intracellular HCV copy numbers did not differ significantly
between ANXA3-knockdown and control cells, indicating equal
viral RNA replication rates (Figure 4D). In contrast, ANXA3
knockdown significantly decreased HCV RNA copy numbers
as well as core protein released in the culture supernatant both
on day 3 and day 6 post-transfection (Figures 4D and 4E). As
HCV RNA copy numbers only poorly reflect the amount of
infectious particles, we determined infectious viral titers by
measuring the 50% tissue culture infective dose (TCID50)
(Lindenbach et al., 2005), using the same experimental setup
as described earlier. Viral titers were severely reduced at day 3
and day 6 post-transfection in ANXA3-knockdown cells in com-
parison to control cells (Figure 4F), supporting the model that
ANXA3 influences HCV particle production.
Core Translocation to Lipid Droplets and CapsidEnvelopment Is Independent of ANXA3No accumulation of infectious viral particles was observed in-
side ANXA3-knockdown cells; instead, we detected slightly
reduced intracellular viral titers pointing to HCV viral assembly
or maturation as the steps in the viral life cycle requiring proper
ANXA3 function (Figure 5A). A prerequisite for HCV assembly is
the trafficking of the capsid protein core and NS5A to lipid drop-
lets. Therefore, we first analyzed the subcellular localization of
the two viral proteins by immunofluorescence microscopy and
lipid droplet isolations. Core and NS5A strongly localized to lipid
droplets in both control and ANXA3-knockdown cells, excluding
that disturbed trafficking of these proteins was responsible for
the defect in virus production observed in ANXA3-knockdown
cells (Figures 5B and 5C). Next, we examined the formation of
high-molecular-mass (HMM) core complexes and envelopment
of the capsids as described previously (Gentzsch et al., 2013).
Cell lysates were analyzed by 2D blue native SDS-PAGE, and
results indicated that formation of HMM core complexes is inde-
pendent of ANXA3 expression for Jc1 aswell as JFH1 strains and
independent on the ANXA3-inactivation method (Figure 5D). For
envelopment assays, cell lysates were treated with proteinase K
to determine the amount of core that is enveloped and, thus,
protected from proteinase K digestion. As a control, lysates
were incubatedwith Triton X-100, which disrupts all membranes.
The amount of core protein that was protected was similar in
control and in ANXA3-knockdown cells, demonstrating that the
envelopment of core was not dependent on ANXA3 expression
(Figure 5E). As control, the membrane was probed with NS5A
antibodies, and, as expected, NS5A is not protected against
proteinase K digestion, similar to core in envelope-deleted viral
strains (Figure 5E). As annexins had been previously described
Jc1ΔE1E2 NS5AB-Fluc
NS4A NS5BLucNS5ANS3Core NS4BNS2p7
A B
D
CEMCVIRES Con1 SGR
NeoRHCVIRES
NS4A NS5BNS5ANS3 NS4B
shN
T
Con
trol
JFH1
Core
NS5A
ANXA3
shA
NX
A3
Tubulin
F
HC
V R
NA
cop
y nu
mbe
r(G
E x
108 /
μg
tota
l RN
A)
day 3 day 6 0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
shNTshANXA3
HC
V R
NA
cop
y nu
mbe
r(G
E x
109
/ ml)
0
0.2
0.4
0.6
0.8
1.0 ** **
day 3 day 6
shNTshANXA3
Infe
ctiv
ity(T
CID
50 x
104 /
ml)
** *
day 3 day 6
shNTshANXA3
0
1
2
3
NS4A NS5BEGFPNS5ANS3Core NS4BE1 E2 NS2p7
Jc1 NS5AB-EGFP
E
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8H
CV
cor
e pr
otei
n(fm
ol x
103
/ ml)
0
** **
day 3 day 6
shNTshANXA3
1
10
100
24 h 48 h 72 h
HC
V R
NA
repl
icat
ion
(RLU
ove
r 4 h
)
shNTshANXA3shNT shANXA3
Intracellular HCV RNA Secreted HCV RNA
Figure 4. ANXA3 Regulates HCV Progeny Virion Production
(A) Huh7.5 cells transduced with lentiviral shRNAs were electroporated with Con1 SGR and kept under selective pressure for 3 weeks, and surviving colonies
were stained with crystal violet. Shown is one representative experiment. shNT, non-targeting shRNA; shANXA3, shRNA targeting ANXA3.
(B) Luciferase activity of Jc1DE1E2NS5AB-Fluc-transfected cells was measured 4, 24, 48, and 72 hr post-electroporation. Shown is the luciferase activity (RLU,
relative light units) per microgram of protein standardized to the 4-hr time point (mean ± SEM, n = 3).
(C) JFH1-electroporated cells were lysed and analyzed by western blotting.
(D–F) Huh7.5 cells transduced with lentiviral shRNAs were electroporated with full-length HCV Jc1NS5AB-EGFP RNA. Equal transfection rates were verified by flow
cytometry of EGFP 3 days post-electroporation. (D) Intra- and extracellular HCV RNA copy numbers were determined by qRT-PCR. Shown is the absolute
quantification of the HCV copy number (GE, genome equivalents) per microgram of total RNA (intracellular) or milliliter of culture supernatant (extracellular)
(mean ± SEM, n = 3). **p < 0.01. (E) Quantification of HCV core protein released in the supernatants (mean ± SEM, n = 3) **p < 0.01. (F) The infectious titers of the
culture supernatants were determined by TCID50 titration on naive Huh7.5 cells (mean ± SEM, n = 3–5). *p < 0.05; **p < 0.01.
to modulate phospholipase A2 (PLA2) activity (Gerke and Moss,
2002) and cPLA2 is required for HCV virion production (Menzel
et al., 2012), we probed cPLA2 activity in HCV-infected control
and ANXA3-knockdown cells and did not detect any difference
(Figure S3).
Knockdown of ANXA3 Affects the Density and SpecificInfectivity of HCV ParticlesTo address whether ANXA3 influences the specific infectivity of
the particles released, we divided the amount of infectious parti-
cles (focus-forming units; FFUs) by the amount of the viral capsid
protein coreor the number ofHCVgenomes. Intriguingly, particles
released by cells lacking ANXA3 had a lower specific infectivity
than the particles released by control cells (Figure 6A). To analyze
whetherANXA3directly influencesparticle infectivity,weanalyzed
whether ANXA3 is incorporated into the virions. Therefore, we
purified E2-FLAG tagged viral particles from culture supernatant
by subsequent precipitation, ultracentrifugation, and affinity puri-
fication, butwedid not detectANXA3attached to thepurifiedHCV
particles (Figure S3). In addition, antibodies directed against
ANXA3 failed to neutralize HCV (Figure S3).
One major determinant of the specific infectivity of HCV parti-
cles is the density of the lipoviroparticles. When we compared
the densities by density gradient centrifugation, we observed
the peak of infectivity and secreted core protein at lower
densities in culture supernatant from cells expressing ANXA3
(Figure 6B); secreted HCV RNA displayed only a slight shift (indi-
cated by arrows). Of note, the higher plateau at lower densities
most likely reflects that there is less viral RNA in ANXA3-knock-
down cell supernatant; thus, this plateau seems higher than in
the control cells when shown as percentage of total RNA. The
change in density during HCV maturation critically depends on
Cell Reports 16, 3219–3231, September 20, 2016 3225
A B C
D
E
Figure 5. Core Trafficking and Capsid Envelopment Is Independent of ANXA3
(A) Equal amounts of HCV Jc1NS5AB-EGFP-electroporated cells were lysed, and intracellular infectivity was determined by TCID50 titration on naive Huh7.5 cells
(mean ± SEM, n = 5). shNT, non-targeting shRNA; shANXA3, shRNA targeting ANXA3.
(B) Huh7.5 cells transduced with lentiviral shRNAswere electroporated with Jc1NS5AB-EGFP-BSD and selected for infected cells with blasticidin. Lipid droplets (LDs)
were isolated by sucrose gradient centrifugation and analyzed by western blotting. The asterisk marks an unspecific band.
(C) Huh7.5 cells transduced with lentiviral shRNAs and infected with Jc1 were stained with core or NS5A antibodies and BODIPY and analyzed by confocal
microscopy (scale bar, 10 mm).
(D) 48 hr post-electroporation of HCV RNA, cell lysates were prepared by freeze-thaw cycles and analyzed by 2D blue native followed by SDS-PAGE andwestern
blotting to detect core in high-molecular-mass (HMM) and low-molecular-mass (LMM) complexes.
(E) Cell lysates were subjected to proteinase K (PK) treatment and analyzed by western blotting. As control, lysates were left untreated or incubated with Triton
X-100 prior to proteolytic digestion. Shown is one representative western blot experiment and the densitometric quantification of core levels as the percentage of
protected core (+PK) compared to untreated control (mean ± SEM, n = 6).
very low-density lipoprotein secretion (Gastaminza et al., 2008;
Huang et al., 2007; Jiang and Luo, 2009). Therefore, we investi-
gated the activity of the microsomal transfer protein (MTP) and
could not detect dependence on ANXA3 in the presence or
absence of HCV replication (Figure 6C). Secretion of apolipopro-
teins ApoE and ApoB into the culture supernatant of ANXA3-
knockdown and control cells was also similar in uninfected cells
(Figure 6D; Figure S4). In contrast, ApoE, but not ApoB, secretion
was significantly reduced in ANXA3-knockdown cells that repli-
cated either subgenomic or genomic HCV RNA (Figure 6D; Fig-
ure S4). Of note, even in HCV-infected cells, the density of total
cellular and secreted ApoE was not affected by ANXA3 knock-
down (Figure 6B; Figure S4). Thus, in uninfected cells, lipoprotein
secretion functions independently from ANXA3, but in HCV-in-
fected cells, ANXA3 contributes to ApoE secretion but not
lipidation.
3226 Cell Reports 16, 3219–3231, September 20, 2016
ANXA3 Promotes Viral Particle Maturation byFacilitating Incorporation of ApoEDuring HCV maturation, ApoE likely attaches to HCV particles
via its interaction with the envelope protein E2 (Lee et al.,
2014). We probed this interaction using a fully infectious HCV
construct expressing FLAG-tagged E2. In cells expressing
ANXA3, we were able to detect the ApoE-E2 interaction. In
contrast, in ANXA3-knockdown or -knockout cells, this interac-
tion was nearly abolished (Figures 7A and 7B). ANXA3 was
additionally required for the interaction between E2 and the
capsid core, implicating that incorporation of the envelope pro-
teins, as well as ApoE, requires ANXA3 (Figures 7A and 7B).
Importantly, the interactions between E2 and ApoE, as well
as core, were only observed in the context of full viral replica-
tion, as co-expressed core and E1/E2 expression constructs
containing the signal peptide of core to ensure correct
A
D
0
4
8
Spe
cific
infe
ctiv
ity(F
FU /
105 G
E)
0
Spe
cific
infe
ctiv
ity(F
FU /
fmol
cor
e)
*
10
20
30
40
50
60
70
80
shNTshANXA3
C
B
0
0.5
1.0
1.5
2.0 shNTshANXA3
- SGR
MTP
act
ivity
(pm
ol tr
ansf
erre
d / μ
g pr
otei
n)
20
40
60
80
100
120
140
0
Apo
E s
ecre
tion
(% o
f con
trol)
Mock SGR
shNT shANXA3
** **
Jc1Jc1
ApoB
ApoB
shN
Tsh
AN
XA
3
ApoE
Sup
.C
ells
TubulinNS5A
ApoE
HC
V R
NA
cop
y nu
mbe
r(%
GE
/ fra
ctio
n)
0
10
20
30
40
1.00 1.04 1.08 1.12 Density
shNTshANXA3
Infe
ctiv
ity(%
TC
ID50
/ fra
ctio
n)
0
10
20
30
40
50
60
1.00 1.04 1.08 1.12 Density
HC
V c
ore
(% /
fract
ion)
-10
102030405060
1.00 1.04 1.08 1.12 Density
00
10
20
30
40
1.00 1.04 1.08 1.12 Density
Apo
E(%
/ fra
ctio
n)
Figure 6. ANXA3 Is Required for Efficient
HCV Maturation
(A) To calculate the specific infectivity of released
HCV particles, infectivity (focus forming units; FFU)
was determined by TCID50 titration and divided by
the amount of HCV core protein or the number of
genome equivalents (GE) (mean ± SEM, n = 3).
*p < 0.05. shNT, non-targeting shRNA; shANXA3,
shRNA targeting ANXA3.
(B) To determine the density of released viral parti-
cles, culture supernatants were analyzed by iodix-
anol gradient centrifugation and viral RNA copy
numbers, infectivity (as TCID50), and core and ApoE
protein levels were determined (mean ± SEM, n = 3).
(C) MTP activity was analyzed in shRNA-transduced
and Con1 SGR-transfected Huh7.5 cells and de-
picted as picomoles of transferred fluorescent
substrate per microgram of protein (mean ± SEM,
n = 10).
(D) Lipoprotein secretion was assessed by western
blotting with ApoE and ApoB antibodies of culture
supernatant (Sup.) of control, Con1 SGR-trans-
fected, or Jc1NS5AB-EGFP-BSD-transfected Huh7.5
cells. Quantification of ApoE secretion by densito-
metric analysis of western blots (mean ± SEM,
n = 3–9). **p < 0.01.
See also Figures S3 and S4.
subcellular localization did not interact with each other or with
endogenous ApoE (Figure 7C). Prior to interaction with E2,
ApoE interacts with NS5A, an interaction that is thought to re-
cruit ApoE to virion assembly sites. We used a viral strain that
encodes an HA-tagged NS5A to study this interaction. Intrigu-
ingly, we detected no change in the level of ApoE interacting
with NS5A in ANXA3-knockdown cells (Figure 7D). Therefore,
ANXA3 is required for virion maturation steps in the ER. To
verify our immunoprecipitation results, we analyzed the sub-
cellular localization of E2/core and ApoE. We observed that,
in uninfected cells, ApoE mainly localizes to the Golgi com-
partment, while in HCV-infected cells, ApoE is scattered in a
punctuate pattern throughout the cell co-localizing with E2 (Fig-
ure 7E). This pattern is reverted in ANXA3-knockdown cells
where ApoE is, again, in the Golgi co-localizing with Golgi ma-
trix protein GM130 (Figure 7F). We observed a similar pattern of
less co-localization in ANXA3-knockdown cells when we
probed for E2 and core (Figure 7E).
Cell Report
In summary, our results suggest that
ANXA3 acts as a host factor for HCV parti-
cle maturation, with subsequent effects on
the number and infectivity of particles
released (Figure 7G).
DISCUSSION
Here, we performed an extensive quanti-
tative lipid droplet proteome analysis of
HCV-infected versus uninfected control
cells to reveal the perturbations caused
by HCV infection and to identify regulators
of HCV replication. We compiled a list of 316 proteins that were
reliably identified in the lipid droplet fractions, with 16 proteins
being recruited to lipid droplets and 21 proteins being dis-
placed from lipid droplets in HCV-infected cells. Strikingly,
some of the recruited proteins are annotated for RNA-binding
proteins, which fits with the present model that part of the
membranous web, where the HCV RNA replicates, as well as
viral translation centers, are found in close proximity of lipid
droplets. Two of our top hits were DEAD box proteins 1 and
3 (DDX1 and DDX3), which both have been described to
localize to lipid droplets in cells expressing the HCV protein
core (Sato et al., 2006), thus validating our SILAC-based
approach. DDX3 was subsequently shown to bind to core
and to interact with the 30UTR of the HCV RNA activating a
non-canonical IkB kinase (IKK)-alpha pathway that, in turn, in-
duces lipogenic genes, resulting in more lipid droplets,
enhanced core-lipid droplet interaction, and virion assembly
(Ariumi et al., 2007; Li et al., 2013).
s 16, 3219–3231, September 20, 2016 3227
A
0.0
0.5
1.0
ApoE(M1)
GM130(M2)
shNTshANXA3
ApoE(M1)
GM130(M2)
***
Jc1 Mock
***M
ande
rs'
colo
c. c
oeffi
cien
ts
*****
0.0
0.5
1.0
ApoE(M1)
Core(M2)
shNTshANXA3
Man
ders
'co
loc.
coe
ffici
ents
0.0
0.5
1.0
Core(M1)
E2(M2)
shNTshANXA3
Man
ders
'co
loc.
coe
ffici
ents *** ***
Man
ders
'co
loc.
coe
ffici
ents
0.0
0.5
1.0
ApoE(M1)
E2(M2)
*** ***
shNTshANXA3
B C
F
G
E
shN
Tsh
AN
XA
3sh
NT
shA
NX
A3
Jc1 FLAG-E2
FLAG-E2ApoECore
Inpu
tIP
:FLA
G FLAG-E2ApoECore*
WT
ΔA
NX
A3
WT
ΔA
NX
A3
Jc1 FLAG-E2
FLAG-E2ApoECoreFLAG-E2ApoECore*
Inpu
tIP
:FLA
G
shN
Tsh
AN
XA
3sh
NT
shA
NX
A3
Jc1 NS5A-HA
NS5A-HAApoEIn
put
IP:H
A NS5A-HAApoE
ApoE/NS5A: 0.5 0.6
FLAG-E2ApoECore
coreE1/FLAG-E2
–+– + +
+––
Inpu
tIP
:FLA
G FLAG-E2ApoECore
D
ANXA3
Entry & Uncoating
Translation & Trafficking
RNA Replication
E2-ApoEMaturation & Release
NS5A-ApoECapsid Assembly
CoreNS5A
EnvelopeProteins
ApoEANXA3ANXA3-independentANXA3-dependent
Core FLAG-E2 Merged
shN
Tsh
AN
XA
3
ApoE FLAG-E2 Merged
shN
Tsh
AN
XA
3
ApoE Core Merged
shN
Tsh
AN
XA
3
ApoE GM130ApoE/GM130
shN
Tsh
AN
XA
3
FLAG-E2
Figure 7. ANXA3 Is Required for Subversion of ApoE Secretion
(A and B) ANXA3-knockdown or -knockout cells were transfected with Jc1Flag-E2, and 5 days post-transfection, cells were lysed in high-stringency buffer for
co-immunoprecipitation (IP) of FLAG-E2 with ApoE and core. shNT, non-targeting shRNA; shANXA3, shRNA targeting ANXA3.
(C) Huh7.5 cells transduced with lentiviral expression constructs for core and E1/FLAG-E2 were lysed in high-stringency buffer for co-immunoprecipitation of
FLAG-E2 with ApoE and core.
(D) shRNA-transduced cells were transfected with Jc1Flag-E2 NS5A-HA RNA and lysed in low-stringency buffer for co-immunoprecipitation of NS5A with ApoE.
(E and F) shRNA-transduced cells were infected with Jc1Flag-E2, fixed, and stained with ApoE, FLAG, core, and GM130 (Golgi) antibodies. Samples were analyzed
by confocal microscopy; single channels are shown in black and white, and the merged images are pseudocolored as indicated (scale bars, 10 mm).
Co-localization was analyzed using coloc2 in Fiji of individual cells of two to three independent experiments by calculating the degree of co-localization using the
Manders’ co-localization coefficient (mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001.
(G) Model: HCV infection causes re-localization of ANXA3 to lipid droplet fractions to ensure efficient HCV maturation and egress.
Proteins depleted from lipid droplets were mainly annotated
for lipid metabolic processes, indicating that HCV perturbs the
protein composition to disconnect the lipid droplets from their
normal metabolic function. This phenomenon might contribute
to the development of steatosis in patients and is line with our
3228 Cell Reports 16, 3219–3231, September 20, 2016
previous data that lipid droplets are stabilized in cells expressing
the capsid protein core (Harris et al., 2011).
One of the proteins highly enriched in lipid droplet fractions
was the phospholipid-binding protein ANXA3 that was studied
in greater molecular detail. This enrichment was not due to an
overall increased expression but due to a specific re-localization
to lipid-rich fractions in HCV-infected cells. As overexpressed
ANXA3 failed to localize to ring-like structures, ANXA3 is likely
trafficked to lipid-rich membrane structures and not to the lipid
droplet surface.We found that expression of single viral proteins,
either core or NS5A, is sufficient to re-localize ANXA3. However,
we were unable to detect a direct interaction by co-immunopre-
cipitation experiments (data not shown) indicating that ANXA3 is
recruited by interaction partners or by changes in the membrane
composition that enable binding of ANXA3. Alternatively, HCV
infection could also cause an efflux of calcium from the ER at
ER-lipid droplet contact sites that, in turn, could enable the
binding of ANXA3 to membranes. Interestingly both core and
NS5A can affect calcium homeostasis in the ER (Dionisio et al.,
2009). Thus, locally elevated calcium levels could trigger
enhanced ANXA3 binding.
We observed that ANXA3 is required for a step in HCV par-
ticle production after envelopment of the viral capsids. Viral
particles released from cells lacking ANXA3 showed a higher
density and less specific infectivity pointing to a defect in
proper virion assembly or maturation. ANXA3 expression was
required for ApoE secretion, but not ApoB secretion or MTP ac-
tivity, in cells infected with HCV but not in uninfected cells.
Interestingly, ApoE subcellular localization changes in response
to HCV infection, a phenotype reverted by ANXA3 knockdown.
Through interaction studies, we show that the recruitment of
ApoE via NS5A to virion assembly sites is independent of
ANXA3, while ApoE/E2 interaction requires ANXA3. Thus,
ANXA3 mediates ApoE trafficking (but not lipidation) and HCV
maturation steps within the ER. Interestingly, it was recently
reported that ANXA3 is secreted and that it functions in a
positive-feedback loop that promotes cancer stem cell self-
renewal and tumor growth in hepatocellular carcinoma (Tong
et al., 2015). ANXA3 expression is approximately 10-fold higher
in cancer cell lines as Huh7 cells; thus, ANXA3 effect on HCV
replication might contribute to the higher replication rates in
Huh7 cells. However, further studies have to explore in greater
detail the mechanistic role of ANXA3 in the late stages of the
HCV life cycle.
EXPERIMENTAL PROCEDURES
For detailed experimental procedures, see the Supplemental Information.
Cell Lines and Culture Conditions
HEK293T cells obtained from the American Type Culture Collection and
Huh7.5 cells obtained from C.M. Rice were grown under standard cell culture
conditions.
HCV Infection and Replicon Assays
HCV Jc1 reporter constructs encoding fluorescent proteins, selection
markers, or the firefly luciferase between a duplicated NS5A-NS5B cleavage
site and Jc1FLAG-E2 were described previously (Eggert et al., 2014; Webster
et al., 2013). For HCV infection and transfection experiments, in-vitro-tran-
scribed HCV RNA was electroporated into Huh7.5 cells. Culture supernatant
was harvested, filtered, and concentrated by polyethylene glycol 8000 precip-
itations. For subgenomic replicon assays, replicon RNA was electroporated
into Huh7.5 cells and viral RNA replication was measured by either survival
under G418 treatment or luciferase activity as described previously (Vogt
et al., 2013).
Knockdown and Knockout of ANXA3
To knock down ANXA3, we used lentiviral shRNA constructs (pSicoR-MS1)
as described previously (Herker et al., 2010; Wissing et al., 2011). We
created clonal ANXA3-knockout cell lines using the CRISPR/Cas9 system as
described previously (Ran et al., 2013).
SILAC Labeling, HCV Infection, and Lipid Droplet Isolation for Mass
Spectrometry
For isotopemetabolic protein labeling, we used the SILACProtein Quantitation
Kit according to the manufacturer’s instructions (Thermo Scientific).
After the incorporation was confirmed, light or heavy Huh7.5 cells were
infected with Jc1 reporter strains and cultured for 2 to 3 weeks. Lipid
droplets were isolated by two sequential sucrose density centrifugations
of post-nuclear supernatant, followed by three washing steps in isotonic
buffer. The lipid-droplet-associated proteins were separated by
SDS-PAGE. After tryptic in-gel digestion, LC-ESI-MS/MS analyses were
performed on a quadrupole-time-of-flight (Q-TOF) mass spectrometer
(Q-TOF Premier, Micromass/Waters) or on a linear trap quadrupole (LTQ)
orbitrap mass spectrometer (Orbitrap Fusion, Thermo Scientific). Both
instruments were coupled with an ESI source to a nano-UPLC (ultra-perfor-
mance liquid chromatography) system (nanoACQUITY, Waters; Dionex
UltiMate 3000 RSLCnano, Thermo Fisher Scientific). Data from LC-ESI-
Q-TOF-MS/MS analysis were analyzed using the open-source software
framework OpenMS (Sturm et al., 2008) and the OpenMS Proteomic
Pipeline (TOPPAS) (Kohlbacher et al., 2007). For peptide and protein iden-
tification, LC-MS/MS raw data were processed as described previously
(Kwiatkowski et al., 2015). SILAC pairs were detected and quantified using
SILACAnalyzer (Nilse et al., 2010). The LC-MS/MS data from orbitrap anal-
ysis were processed with MaxQuant version 1.5.2.8 (Cox and Mann, 2008).
The MS proteomics data have been deposited into the ProteomeXchange
Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE
partner repository (Vizcaıno et al., 2013) with the dataset identifier
PXD004707.
Biochemical and Cell Biological Methods
Lipid droplets were isolated by sucrose gradient centrifugation (Herker et al.,
2010). Iodixanol gradient centrifugations for membrane flotation and to deter-
mine HCV particle density were performed as described previously (Catanese
et al., 2013; Vogt et al., 2013). 2D blue native SDS-PAGE and proteinase
K digestion protection assay were performed as described (Gentzsch et al.,
2013). Co-immunoprecipitation, determination of ApoE/B secretion, and
immunofluorescence microscopy were performed as described previously
(Herker et al., 2010).
Bioinformatics and Statistical Analysis
For bioinformatics and statistical analysis, we used R (R Core Team, 2015),
RStudio (RStudio Team, 2015), and GraphPadPrism (GraphPad Software).
Statistical analysis was performed using unpaired two-tailed Student’s t test
and, in the case of normalized data, one-sample t test.
ACCESSION NUMBERS
The accession number for the data reported in this paper is PRIDE:
PXD004707.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2016.08.052.
AUTHOR CONTRIBUTIONS
K.R. designed, conducted, and analyzed the experiments. M.K., M.W., and H.S.
performed the MS experiments for proteome analysis and analyzed the data.
S.H., A.S., and C.G. provided reagents and performed some experiments. E.H.
Cell Reports 16, 3219–3231, September 20, 2016 3229
designed and analyzed the experiments and supervised the project. The manu-
script was written by E.H. and K.R., with input from all authors.
ACKNOWLEDGMENTS
We thank R. Bartenschlager (University of Heidelberg) for Jc1 constructs; C.M.
Rice (Rockefeller University) for Huh7.5 cells and RFP-NLS-IPS;
J. McLauchlan (Medical Research Council Virology Unit) for the JFH1
construct; T. Wakita (National Institute of Infectious Diseases, Japan) for
JFH1; B. Webster and W.C. Greene (Gladstone Institute of Virology and
Immunology) for the HCVcc reporter constructs; J.H. Ou (University of
Southern California) for the pUC Con1 subgenomic replicon; M. Spindler for
pSicoR-MS1; F. Zhang (Broad Institute) for pSpCas9 (BB)-2A-Puro (PX459,
Addgene plasmid # 48139); B. Fehse (University Clinic Hamburg Eppendorf)
for LeGO vectors; and C. Thiele (University of Bonn) for LD540. This work
was supported by funds from the DFG (HE 6889/2-1 to E.H. and INST
337/15-1 2013 and INST 337/16-1 2013 to H.S.). The Heinrich Pette Institute,
Leibniz Institute for Experimental Virology, is supported by the Free and
Hanseatic City of Hamburg and the Federal Ministry of Health. The funders
had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Received: November 12, 2015
Revised: May 20, 2016
Accepted: August 16, 2016
Published: September 20, 2016
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