Quantitative Lipid Droplet Proteome Analysis Identifies ... · Cell Reports Article Quantitative...

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University of Groningen Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production Roesch, Kathrin; Kwiatkowski, Marcel; Hofmann, Sarah; Schoebel, Anja; Gruettner, Cordula; Wurlitzer, Marcus; Schlueter, Hartmut; Herker, Eva Published in: Cell reports DOI: 10.1016/j.celrep.2016.08.052 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database 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 Particle Production. Cell reports, 16(12), 3219-3231. https://doi.org/10.1016/j.celrep.2016.08.052 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 25-03-2020

Transcript of Quantitative Lipid Droplet Proteome Analysis Identifies ... · Cell Reports Article Quantitative...

Page 1: Quantitative Lipid Droplet Proteome Analysis Identifies ... · Cell Reports Article Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle

University of Groningen

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

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2016

Link to publication in University of Groningen/UMCG research database

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

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 25-03-2020

Page 2: Quantitative Lipid Droplet Proteome Analysis Identifies ... · Cell Reports Article Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle

Article

Quantitative Lipid Droplet

Proteome AnalysisIdentifies Annexin A3 as a Cofactor for HCV ParticleProduction

Graphical 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

[email protected]

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

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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/).

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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,

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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

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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

AN

XA

3W

T

Tubulin

ANXA3

Par

enta

l CRISPR Clones

*

ΔANXA3 WT

#1 #2 #3 #4

H

I J

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AN

XA

exp

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ion

0 0.2 0.4 0.6 0.8 1.0 1.2

AN

XA

2 A

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XA

4 A

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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

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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

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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

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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

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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

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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

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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

Page 13: Quantitative Lipid Droplet Proteome Analysis Identifies ... · Cell Reports Article Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle

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

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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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