Palmitoylation of Hepatitis C Virus Core Protein Is Important for Virion Production

Abstract

Hepatitis C virus core protein is the viral nucleocapsid of hepatitis C virus. Interaction of core with cellular membranes
like endoplasmic reticulum (ER) and lipid droplets (LD) appears to be involved in viral assembly. However, how these interactions
with different cellular membranes are regulated is not well understood. In this study, we investigated how palmitoylation,
a post-translational protein modification, can modulate the targeting of core to cellular membranes. We show that core is
palmitoylated at cysteine 172, which is adjacent to the transmembrane domain at the C-terminal end of core. Site-specific
mutagenesis of residue Cys172 showed that palmitoylation is not involved in the maturation process carried out by the signal peptide peptidase or in the
targeting of core to LD. However, palmitoylation was shown to be important for core association with smooth ER membranes and
ER closely surrounding LDs. Finally, we demonstrate that mutation of residue Cys172 in the J6/JFH1 virus genome clearly impairs virion production.

Full text

Palmitoylation of Hepatitis C Virus Core Protein Is Important
for Virion Production
*
□
S
Received for publication, May 7, 2009, and in revised form, September 11, 2009 Published, JBC Papers in Press, September 25, 2009, DOI 10.1074/jbc.M109.018549
Nathalie Majeau, Re´mi Fromentin, Christian Savard, Marie Duval, Michel J. Tremblay, and Denis Leclerc
1
From the Infectious Disease Research Centre, CHUL, Universite´ Laval, 2705 boulevard Laurier, Que´bec G1V 4G2, Canada
Hepatitis C virus core protein is the viral nucleocapsid of hep-
atitis C virus. Interaction of core with cellular membranes like
endoplasmic reticulum (ER) and lipid droplets (LD) appears to
be involved in viral assembly. However, how these interactions
with different cellular membranes are regulated is not well
understood. In this study, we investigated how palmitoylation, a
post-translational protein modification, can modulate the tar-
geting of core to cellular membranes. We show that core is pal-
mitoylated at cysteine 172, which is adjacent to the transmem-
brane domain at the C-terminal end of core. Site-specific
mutagenesis of residue Cys
172
showed that palmitoylation is not
involved in the maturation process carried out by the signal pep-
tide peptidase or in the targeting of core to LD. However, palmi-
toylation was shown to be important for core association with
smooth ER membranes and ER closely surrounding LDs. Finally,
we demonstrate that mutation of residue Cys
172
in the J6/JFH1
virus genome clearly impairs virion production.
Hepatitis C virus (HCV)
2
is a major causative agent of
chronic hepatitis (1). HCV is an RNA virus of the Flaviviridae
family and has a single-stranded, positive sense RNA genome of
9.6 kb (2). The HCV RNA genome encodes a polyprotein of
!3000 amino acids (aa) that is processed by host and viral pro-
teases into 10 different components (3). Core protein is the only
virus-encoded nucleocapsid protein involved in assembly and
packaging of the viral plus-strand RNA genome (3). The C-ter-
minal signal sequence (aa 173–191) facilitates channeling of the
nascent HCV polyprotein to the endoplasmic reticulum (ER)
(4). After cleavage, core protein (191 aa) is released and further
processed by an intramembrane protease, the signal peptide
peptidase (spp), to yield a protein of 177 aa (5, 6). The fully
processed core protein interacts mainly with lipid droplets (LD)
and ER membranes and was also reported to be translocated
into the nucleus (7–9). The C-terminal part of core (aa 120–
191) includes a predicted amphipathic
!
-helix that is responsi-
ble for core association with LD and ER membranes (8, 10).
Recent studies have indicated that assembly of HCV particles
occurs on ER membranes that are associated closely with LD
(11). Core protein on LD recruits the viral proteins of the rep-
lication complex and is translocated to ER-associated mem-
branes where it interacts with HCV RNA to produce assembled
viral particles (11). To facilitate HCV assembly, core protein
also promotes LD accumulation when expressed in cells (12,
13). HCV core protein as well as the replication complex are
also found in the detergent-resistant membrane (DRM) frac-
tion, which is distinct from the classical lipid rafts (14–16).
Because HCV core is targeted to different organelle mem-
branes during the viral life cycle, we investigated whether post-
translational modification of core in the form of palmitoylation
could be involved in this trafficking. Palmitoylation or S-acyla-
tion is the covalent attachment of a fatty acid group, usually the
saturated 16 carbon palmitate to cysteine residues via a thio-
ester bond (17). Protein palmitoylation enhances surface hy-
drophobicity and membrane affinity and plays an important
role in modulating protein trafficking (18).
In this study, we identified a site for palmitoylation in HCV
core protein. The cysteine modified by a palmitate moiety is
adjacent to the ER-targeting domain located at the C terminus.
We demonstrate that spp processing and LD targeting was
unaffected by impairment of this palmitoylation generated by
mutation. However, mutated HCV core protein accumulated
differently in the ER membrane and was poorly associated with
ER surrounding LD. It is of high importance that we observed
that a mutation in the palmitoylation site of HCV core impaired
viral infectivity.
EXPERIMENTAL PROCEDURES
Construction of pPIC3.5Kcore, pcDNA3.1core, and J6/JFH1
Mutants—Clones of Pichia pastoris expressing HCV core C (aa
1–191) of strain H77 (genotype 1a) and spp, a signal peptide
peptidase of human cell origin, have been described previously
(19). Mutations were introduced by PCR and conventional
cloning methods. Cys
172
and Cys
91
were replaced by Ser and
Leu, respectively. Yeast cells were transformed, and protein
expression was induced with methanol as described previously
(20). The coding sequences of the HCV core were inserted into
the polylinker site of pcDNA3.1 (Invitrogen) with BamHI and
EcoRI restriction sites (New England BioLabs). Vaccinia virus
expressing HCV core-E1 protein (Sc59 6C/Ss) was kindly pro-
vided by Chiron (Emeryville, CA). Vaccinia Ankara strain ex-
pressing T7 polymerase was generously provided by Bernard
Moss (NIAID, National Institutes of Health, Bethesda, MD).
The plasmid FL-J6/JFH-5"C19Rluc2AUbi, which consists of
the full-length HCV genome and expresses Renilla luciferase,
*This work was supported by grants from the Canadian Institutes of Health
Research of Canada and the Re´seau Sida et Maladies Infectueuses of the
Fonds de la Recherche en Sante´ du Que´bec.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1.
1
To whom correspondence should be addressed. Tel.: 418-654-2705; Fax:
418-654-2715; E-mail: denis.leclerc@crchul.ulaval.ca.
2
The abbreviations used are: HCV, hepatitis C virus; ER, endoplasmic reticu-
lum; LD, lipid droplets; spp, signal peptide peptidase; aa, amino acid(s);
DRM, detergent-resistant membrane; PBS, phosphate-buffered saline;
2-BP, 2-bromopalmitate; wt, wild type; NLP, nucleocapsid-like particle;
TMD, transmembrane domain.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 49, pp. 33915–33925, December 4, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
DECEMBER 4, 2009•VOLUME 284• NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33915
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was kindly provided by Charles M. Rice (The Rockefeller Uni-
versity, New York, NY). The substitution of Cys for Ser in J6
core protein was introduced by PCR and cloned into the
J6/JFH1 clone as a BglII/BsiWI fragment. All of the plasmid
HCV sequences were verified by sequencing.
Biotin Switch Assay to Detect Palmitoylation—Palmitoyla-
tion of core was examined by a recently developed biotin switch
assay technique (21). In this protocol, acylation groups attached
to cysteine residues via thioester bonds are replaced with biotin
moieties. Yeast cell cultures (200 ml) expressing spp or co-ex-
pressing spp and core proteins were induced as previously
described (20). The cells were collected and ground to a fine
powder in liquid nitrogen. For analysis of human hepatoma
cells, Huh7.5 cells were infected with vaccinia virus (C-E1).
After 24 h, the cells were washed with phosphate-buffered
saline (PBS) and harvested using a rubber policeman. The cells
were recovered by centrifugation and frozen at #80 °C. The
samples were resuspended in lysis buffer (150 mMNaCl, 5 mM
EDTA, 1$complete protease inhibitors (Roche Applied
Science), 50 mMTris, pH 7.4) containing 10 mMN-ethylmale-
imide (Pierce) and 1.7% Triton X-100. The samples were incu-
bated for1hat4°Cfollowed by centrifugation at 4 °C at 500 $
gto remove insoluble material. The proteins were precipitated
with methanol/chloroform, and the air-dried pellet was resus-
pended in 3.6 ml of SDS buffer (1% SDS, 100 mMNaCl, 0.2%
Triton X-100, 50 mMTris"HCl, pH 7.4) and incubated over-
night at 4 °C with 5 mMN-ethylmaleimide. The proteins were
precipitated three times with methanol/chloroform and resus-
pended in 1.5 ml of SDS buffer to be further divided into two
equal aliquots. One aliquot was combined with 2.9 ml of 0.7 M
fresh hydroxylamine, 1$complete protease inhibitors, 0.2%
Triton X-100, and 1 mMbiotin-N-(6-(biotinamido)hexyl)-3"-
(2"-pyridyldithio)-propionamide (HPDP) (Thermo Scientific).
As a control, the remaining aliquot was treated using the
same procedure, but hydroxylamine was replaced with 50
mMTris, pH 7.4. Both samples were incubated at room tem-
perature for 1 h. The proteins were precipitated using meth-
anol and chloroform and resuspended in 1.2 ml of biotin
buffer (0.4% SDS, 120 mMNaCl, 0.16% Triton X-100, 5 mM
EDTA, 0.16 mMbiotin-HPDP 1$inhibitor mixture, 50 mM
Tris"HCl, pH 7.4) and incubated for1hatroom temperature.
The proteins were precipitated three times using the same pro-
cedure and resuspended in 2.5 ml of lysis buffer containing 0.2%
Triton X-100 and 0.1% SDS. The samples were centrifuged at
15,000 $gfor 1 min, and aliquots were removed as controls.
The remaining reactions were incubated with 15
"
l of neutra-
vidin-agarose beads (Thermo Scientific) at room temperature
for 90 min. The beads were washed four times with lysis buffer
containing 0.2% Triton X-100 and 0.1% SDS, and the proteins
were eluted by adding 1%
#
-mercaptoethanol to the washing
buffer and incubating for 15 min at 37 °C. The samples were
centrifuged at 15,000 $gfor 1 min, and aliquots were removed
as controls (22).
[
3
H]Palmitic Acid Labeling—Yeast cultures were labeled with
[
3
H]palmitic acid as previously described (23). P. pastoris was
cultured overnight in 200 ml of minimal glucose medium
(Invitrogen) at 30 °C to an optical density of 2 and, after a short
centrifugation, transferred to 20 ml of minimal glucose me-
dium %0.1% methanol (Invitrogen) for6hinthepresence of
the fatty acid synthesis inhibitor cerulenin (2 mg/ml). In some
cell cultures, 50
"
M2-bromopalmitate (2-BP) or Me
2
SO were
also added. The cell cultures were then incubated for 2 h with 1
mCi of [9,10-
3
H(N)]palmitic acid (Amersham Biosciences) (50
Ci/mmol). The cells were disrupted with sarkosyl (0.5%), and
the proteins were denatured with 1% SDS. The protein samples
were resolved on nonreducing 10% SDS-PAGE. The gel was
blotted onto a nitrocellulose filter and exposed to a [
3
H] inten-
sifying screen for 7 days. The signals were revealed using phos-
phorimaging. The blot was then analyzed further by Western
blotting using anti-core antibodies 537 (19). The Huh7.5 cells
labeling with [
3
H]palmitic acid was performed as previously
described (24). The cells were infected with vaccinia virus and
vaccinia virus expressing HCV core protein and incubated
overnight in medium (Dulbecco’s modified Eagle’s medium, 2%
fetal bovine serum, nonessential amino acids, 5 mMsodium
pyruvate) supplemented with 37
"
Ci/ml of [9,10-
3
H(N)]-
palmitic acid (PerkinElmer). The cells were washed with PBS
and lysed in radioimmune precipitation assay buffer (50 mM
Tris"HCl, pH 7.5, 150 mMNaCl, 5 mMEDTA, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1$complete protease
inhibitors). The cell lysate was resolved in a nonreducing SDS-
PAGE and analyzed with phosphorimaging.
Flotation Assay—The flotation assay was carried out as
previously described (25). Two hours after infection of
Huh7.5 cells with vaccinia virus carrying the T7 polymerase
gene, the cells were transfected with pcDNA3.1 constructs
using FuGENE 6 transfection reagent (Roche Applied Science).
In some cases, 50
"
Mof 2-BP was added to the medium. Twen-
ty-four hours after transfection, the cells were washed with ice-
cold PBS and then harvested using a rubber policeman. The
collected cells were suspended in 0.6 ml of TNEi buffer (150 mM
NaCl, 2 mMEDTA, 1$complete protease inhibitors, 50 mM
Tris, pH 7.4), homogenized with a Dounce homogenizer and
then resuspended using a 25-gauge needle. The samples were
split into two equal portions, and each was incubated for 30 min
on ice with or without 1% Triton X-100. The lysates were mixed
with 0.6 ml of Optiprep (Sigma) to a final concentration of 40%
iodixanol. This mixture was overlaid with 2.9 ml of 30% iodixa-
nol and 400
"
l of TNEi and then centrifuged at 40,000 rpm, 4 °C
for 4 h in a SW60ti rotor (Beckman Coulter, Fullerton, CA). The
fractions (0.4 ml) were collected from the top of the centrifug-
ing tube and then precipitated with 4 volumes of cold acetone.
The pellets were resuspended in loading buffer, boiled, and sub-
jected to SDS-PAGE and Western blotting. The proteins were
revealed with anti-core, anti-caveolin-1 (Sigma), and anti-cal-
nexin (Sigma) antibodies.
Immunofluorescence Microscopy—Huh7.5 cells were trans-
fected with pcDNA3.1 plasmids using FuGENE 6 transfection
reagent and grown on glass coverslips. Two days after transfec-
tion, the cells were fixed with 4% paraformaldehyde in PBS for
20 min at room temperature followed by 15 min in 0.1% Triton
X-100 in PBS. Primary antibodies (anti-core 537) were diluted
in 5% bovine serum albumin and incubated with cells for2hat
room temperature. After three washes in PBS, Alexa fluor 488
goat anti-rabbit IgG (Invitrogen) were added to cells at a 1:200
dilution for 1 h at room temperature. To stain lipids, the slides
Palmitoylation of HCV Core
33916 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 49• DECEMBER 4, 2009
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were rinsed in 60% isopropanol, incubated in 5 mg/ml oil red O
(Fisher Scientific, Pittsburgh, PA), in 60% isopropanol for 2 min
at room temperature, and rinsed in 60% isopropanol again.
After staining, the slides were washed in PBS and mounted in
ProLong Antifade (Molecular Probes). Microscopy was per-
formed using a Zeiss confocal laser scanning microscope LSM
510, and the images were captured with a Nikon Eclipse TE 300.
Image analysis of LD content was performed using Cell profiler
software (26) on 35 micrographs for each sample. The area
occupied by LD was evaluated for each positive cell.
ER Extraction and Fractionation—For extraction of ER
membranes, Huh7.5 cells infected with vaccinia T7 polymerase
and transfected for 24 h with pCDNA3.1 expressing core or
core mutant C172S were washed with ice-cold phosphate-buff-
ered saline (PBS), scraped into homogenization buffer (250 mM
sucrose, 1 mMEDTA, 1$complete protease inhibitors, 10 mM
Hepes-NaOH, pH 7.4) to be disrupted by Dounce homogeniza-
tion and repeated passages through a fine syringe needle. The
extract was spun down at 1,500 $gfor 10 min, and the super-
natant was further centrifuged at 150,000 $gfor 1 h in a 70.1ti
rotor (Beckman Coulter, Fullerton, CA). The resulting pellet,
representing the membrane fraction, was resuspended in
homogenization buffer and layered (0.5 ml) on top of an iodixa-
nol step gradient composed of 0.5 ml of 10%, 0.5 ml of 15%, 1 ml
of 20%, 0.5 ml of 25%, and 1 ml of 30% iodixanol and centrifuged
at 200,000 $gat 4 °C for 2.5 h in a SW60Ti rotor (Beckman
Coulter). Fractions (0.5 ml) were collected from top to bottom,
and the fraction proteins were analyzed by SDS-PAGE and
immunoblotting using anti-core 537 and anti-calnexin (Sigma)
antibodies.
Immunoelectron Microscopy—Yeast cells were fixed as de-
scribed in Ref. 27 with 3% paraformaldehyde containing 0.2%
glutaraldehyde in 0.1 Mphosphate buffer, pH 7.2, at room tem-
perature for 2 h and then washed well with 0.1 Mphosphate
buffer, pH 7.2. Fixed cells were treated with 1% metaperiodate
for 30 min followed by 30 min of incubation with 50 mM
NH
4
Cl/PO
4
, pH 7.2. After washing, the cells were embedded in
LR white. Ultrathin sections were incubated with anti-core 537
(1:2000) and then with gold-labeled anti-rabbit IgG (Amer-
sham Biosciences). The ultrathin sections were stained with
uranylacetate and lead citrate and analyzed with a JEOL 1010
80-kV transmission electron microscope. Image analysis of the
LD content was performed with cell profiler software (26) on
20 micrographs taken at low magnification (10,000$and
12,000$), which included over 300 cells for each clone. The
area occupied by LD on the micrograph was normalized to the
area occupied by all the cells on the same micrograph.
Production of Infectious HCV and Infection of Huh7.5 Cells—
The plasmid FL-J6/JFH-5"C19Rluc2AUbi wt and mutated
(C172S) were linearized with XbaI and treated with mung bean
nuclease (New England Biolabs) to yield the exact HCV 3"end
(28). In vitro transcription was performed with a T7 RiboMAX
express large scale RNA production system (Promega) follow-
ing the manufacturer’s instructions. Subconfluent Huh7.5 cells
were trypsinized, washed twice with ice-cold OptiMEM
(Invitrogen), and resuspended in OptiMEM at 10 $10
6
/ml.
Four hundred microliter of cells were mixed with 10
"
gofin
vitro transcribed RNA or not (mock) in a cuvette with a gap
width of 0.4 cm (Bio-Rad) and immediately pulsed at 960 farads
and 270 V using a Gene Pulser system (Bio-Rad). Electropo-
rated cells were transferred in 4 ml of complete medium, and
300
"
l were cultured for 4 or 72 h in a 24-well plate prior to be
lysed in Renilla lysis buffer (Promega). For infection, 5 $10
4
naïve Huh7.5 cells, cultured in 24-well plate, were inoculated
overnight by 250
"
l of filtered supernatant harvested 3 days
post-electroporation. After being washed twice in PBS, infected
cells were left in culture for 48 h additional and lysed in Renilla
lysis buffer. Luciferase activity was measured for 10 s, using a
luminometer.
Quantitative Detection of HCV RNA by Quantitative Re-
verse Transcription-PCR—Viral RNA was isolated from 50
"
l of supernatants harvested 3 days post-electroporation
using the MagMAX-96 viral RNA isolation kit as recom-
mended by the manufacturer (Ambion, Austin, TX). 7.5
"
l
of sample were used for quantitative reverse transcription-
PCR analysis employing an Applied Biosystems 7500 sequence
detection system (Applied Biosystems, Foster City, CA).
Amplifications were conducted in duplicate with the TaqMan
RNA-to-Ct 1 step as described by the manufacturer (Applied
Biosystems) using 0.5
"
Mof primers amplified a conserved
segment of the 5"-untranslated region of HCV genotype 2a
(reverse, 5"-GAGTGGGTTTATCCAAGAAAG-3", and for-
ward, 5"-TCTGCGGAACCGGTGAGT-3") and 0.2
"
Mof the
TaqMan probe HCV 2a (5"-FAM-CCGGAATTGCCGGG-
AAGACTG-BHQ
-1
-3") (Biosearch Technologies, Novato,
CA). The amounts of HCV RNA were calculated by compar-
ison with serially diluted in vitro transcripts purified as
described above. This assay was linear between 10
7
and 10
1
RNA copies/microliter.
Statistical Analysis—For the data depicted in Figs. 9 and 11,
statistical significance between groups was determined by anal-
ysis of variance. Calculations were made with Prism version
3.03 software. pvalues &0.05 were considered statistically sig-
nificant. The statistical significance of the results was defined
by performing a one-way analysis of variance combined with
Turkey’s post tests to compare all pairs of columns.
RESULTS
HCV Core Is Palmitoylated—Upon cleavage of HCV poly-
protein by signal peptidase, HCV core stays anchored to ER
membranes through its C-terminal transmembrane domain. A
subsequent maturation event of core meditated by spp cleaves
this hydrophobic domain to produce the mature core, which is
predicted by algorithm for amphiphilicity index (29) to be a
soluble protein. However, most of the processed core remains
tightly associated to intracellular membranes (30). This associ-
ation depends on the integrity of an amphipathic
!
-helix span-
ning aa 116–134 (8). In this study, we investigated whether the
affinity of core to membranes was enhanced by post-transla-
tional modification such as S-acylation. Although there is no
unique canonical motif for acylation sites, the computer algo-
rithm CSS-Palm 2.0 predicts potential palmitoylation sites by
clustering and scoring known palmitoylated proteins (31). This
algorithm was applied to the sequence of the mature HCV core
protein genotype 1, which contains three cysteine residues (i.e.
Cys
91
, Cys
128
, and Cys
172
). The program highlights a possible
Palmitoylation of HCV Core
DECEMBER 4, 2009•VOLUME 284• NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 33917
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palmitoylation site at Cys
172
using a threshold for prediction
corresponding to 89% accuracy and 94% specificity (Table 1).
The palmitoylation site is predicted only for the 177-aa core
that has been processed by spp. Palmitoylation of Cys
172
is not
predicted in the context of the HCV polyprotein, when core
protein is still fused to E1, or in its immature form comprising
191 aa that is generated upon signal peptidase cleavage.
To determine whether HCV core protein is indeed modified
by the addition of a lipid moiety (e.g. palmitoylation), we mea-
sured its endogenous acylation state using the biotin switch
assay. This method relies on the exchange of thioester-linked
protein acyl modifications for biotin moieties (21). HCV core
(191 aa) was co-expressed with human spp and extracted from
P. pastoris as described previously (20). As a first step, free sulf-
hydryl groups of proteins were blocked by incubation with
N-ethylmaleimide. Then fatty acid groups were specifically
removed using hydroxylamine to generate a free cysteine resi-
due at the palmitoylation site. The newly exposed sulfhydryl
groups were labeled with thiol-reactive biotin. The proteins
were then precipitated with neutravidin and analyzed by West-
ern blotting. Incomplete blockage of the cysteine residue prior
to acyl group exchange could lead to false positive results. To
exclude this possibility, hydroxylamine was omitted and
replaced by Tris in half the samples. These control samples
should give negative signals if the protein has been adequately
blocked by N-ethylmaleimide. As shown in Fig. 1A, biotinylated
core protein was present in the hydroxylamine-treated sample
and absent from the nonhydroxylamine-treated sample. Prior
to extraction, yeast cell samples were also incubated for 24 h
with the palmitoylation inhibitor 2-BP. As shown in Fig. 1A, for
the same amount of initial core protein, 2-BP reduced the
amount of biotinylated protein recovered in the neutravidin-
purified samples in a dose-dependent manner.
To verify whether this modification occurs also in human
cells, the biotin switch assay was performed on protein extracts
of hepatocytes expressing HCV core (Fig. 1B). Huh7.5 cells
were infected with vaccinia virus expressing HCV protein from
1 to 382 aa (C-E1). In this construct, the core protein is cleaved
first with signal peptidase followed by spp maturation. Bio-
tinylated core protein was present in the protein sample treated
with hydroxylamine and extracted with neutravidin. These
results indicate that HCV core protein is also an acylated pro-
tein in Huh7.5 cells.
To confirm the observations made with the biotin switch
experiments, we performed a metabolic labeling assay with
radioactive palmitate. P. pastoris expressing spp alone or co-
expressing core and spp were grown in minimal medium sup-
plemented with [
3
H]palmitate for 2 h. As well, Huh7.5 cells
expressing core protein were also incubated with [
3
H]palmitate
for 24 h. The proteins were extracted and resolved on SDS-
PAGE under nonreducing conditions. A radioactive band of 21
kDa was present in cells expressing core but absent in mock
transfected cells or cells incubated with 2-BP and that for yeast
extracts (Fig. 2A) or the hepatocytes samples (Fig. 2B). Western
blot analysis of these samples revealed that these protein bands
corresponded to core protein (data not shown). These results
confirmed that HCV core is indeed a palmitoylated protein.
Identification of Cysteine Residue Involved in HCV Core
Palmitoylation—Cysteines Cys
128
and Cys
172
in core protein
are well conserved among the sequences of different HCV gen-
otypes. Only a JFH1 strain of genotype 2 shows a phenylalanine
at position 172. Cys
91
is not conserved in the core sequences of
the different genotypes and is often replaced by Leu or Met
(supplemental Table S1). To identify the cysteine involved in
palmitoylation of core, we mutated Cys
91
or Cys
172
to Leu or
Ser, respectively, to abolish putative acylation reaction at these
sites. Residue Cys
128
was mutated to Ala or Ser. Mutations of
Cys
128
generated a protein that was unstable in both P. pastoris
and human cells and barely detectable by Western blot. There-
fore, we excluded the Cys
128
mutated clones from our investi-
gation. The biotin switch test was
performed on P. pastoris cells ex-
pressing core mutants. As seen in
Fig. 3, C172S mutations reduced the
amount of biotinylated core present
in the neutravidin-purified samples,
although the amount of core protein
was essentially the same in all of the
samples before affinity extraction.
Mutated protein C91L reacted dif-
ferently from the C172S mutation;
the level of protein purified from
the biotinylated C91L sample was
similar to that of the wt protein
sample. These results suggest that
Cys
172
of core is the major site of
palmitoylation.
FIGURE 1. Detection of acylation of the HCV core protein by biotin switch assay. A,totalcelllysatesfromyeast
P. pastoris expressing spp (mock)orexpressingsppandcore were prepared and treated with N-ethylmaleimide to
block free cysteines. The protein extracts were split in half and treated (%) or not (#) with hydroxylamine
(NH
2
OH) to remove lipid moieties. The newly exposed cysteine residues were then biotinylated with biotin-
HPDP. The biotinylated proteins (acylated proteins) were precipitated with neutravidin-agarose beads for
subsequent immunoblotting analysis with anti-core antibodies. An inhibitor of palmitoylation, 2-BP (I), was
added to the growth medium during induction at final concentrations of 100 (I
100
) or 400 (I
400
)
"
M.%and #
above the lanes indicate hydroxylamine-treated (%) or untreated (#) protein, and neutravidin precipitation
pellet (%) or total extract (#). B, Huh7.5 cells were infected with vaccinia virus alone (mock) or with vaccinia
virus expressing HCV core protein and harvested after 24 h. The proteins were extracted and subjected to the
biotin switch assay as described for A. Acylated proteins were analyzed by Western blot using anti-core
antibodies.
TABLE 1
Palmitoylation site prediction in HCV core protein
The amino sequence of the mature core protein was analyzed for palmitoylation site
probability using the updated software CSS-Palm (28). Score 1 was the result
obtained with core protein in the context of the HCV polyprotein (unprocessed core
protein (191 aa) gave the same results; not shown). Score 2 reflects the result for the
form of core protein processed by spp. The cut-off with low threshold is 0.6 (accu-
racy, 76%; specificity, 75%) and with high threshold is 1 (accuracy, 89%; specificity,
94%).
Position Peptide Score 1 Score 2
91 NEGCGWA 0.139 0.139
128 TLTCGFA 0.052 0.052
172 LPGCSFS 0.417 1.122
a
a
Only Cys
172
in the processed form of core has a score indicating a high likelihood
of palmitoylation.
Palmitoylation of HCV Core
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Effect of Cys
172
Mutation on spp Cleavage—Processing of
HCV core protein by spp is regulated by the C terminus of the
core as well as by other internal sequences known to stabilize
the protein at ER membranes (32, 33). To determine whether
mutation of Cys
172
could affect processing of core by spp, we
expressed these proteins in P. pastoris in the presence or
absence of human spp expression. In these cells, maturation of
core by the resident yeast spp has previously been shown to be
inefficient, but maturation of core can be brought to comple-
tion when the human spp is co-expressed with HCV core (20).
As shown in Fig. 4A, wt core protein extracted from yeast co-
expressing human spp migrated faster than the immature form.
When residue Cys
172
was mutated to Ser, similar results were
obtained, suggesting that maturation of core was not influenced
by palmitoylation of the protein. To confirm this result in
human hepatocytes where resident spp can cleave efficiently
core protein, we analyzed maturation of our constructs in the
presence or absence of (Z-LL)
2
-ketone, an spp inhibitor. We
observed that C172S mutant proteins migrated similarly to wt
protein; unprocessed protein was not detected with the core
protein sample in the absence of the inhibitor (Fig. 4B). From
these results we concluded that palmitoylation of core at Cys
172
is not required for maturation by spp.
Effect of Cys
172
Mutation on Targeting to DRMs—Recent
studies have shown that HCV core is associated with DRMs or
lipid rafts (6, 14, 30). DRM targeting has been recognized as one
of the main functions of palmitoylation of proteins (18). DRMs
are specialized membrane subdomains that are resistant to sol-
ubilization by cold nonionic detergents such as Triton X-100
(25). Therefore, we examined whether palmitoylation is re-
sponsible for localization of the HCV core protein to DRM.
Wild type or mutated forms of C172S core proteins were
expressed in Huh7.5 cells, solubilized at 4 °C in the presence or
absence of 1% Triton X-100, and subjected to a flotation cen-
trifugation assay. The fractions were collected from the top of
the tube and analyzed by Western blotting. In the absence of
detergent, the majority of the wild type and the mutated core
were found in the membrane-containing fractions (fractions 2
and 3) (Fig. 5A). When treated with Triton X-100, a consider-
able amount of wt core protein remained associated with the
DRMs (fraction 2), whereas the rest migrated to the bottom of
the gradient, which corresponded to the detergent-soluble frac-
tion (Fig. 5B). As markers for lipid rafts and ER, we identified
the location in the gradient of caveolin-1 and calnexin by West-
ern blot using specific antibodies on sample taken from the
flotation assay (Fig. 5B). The pattern
of the mutated protein C172S in the
flotation assay was similar to that of
wt core. C172S mutant core protein
was also detected with DRMs and in
the detergent-soluble fraction after
Triton extraction. We concluded
that palmitoylation of the Cys
172
residue is not essential for associa-
tion of the core to DRMs.
Effect of Cys
172
Mutation on HCV
CoreCellularLocalization—Inhepa-
tocytes, core is localized mainly on
the surface of LD and on the ER sur-
rounding LD (11). To examine the
effect of palmitoylation of HCV core
on LD association, Huh7.5 cells
were transfected with plasmid
pcDNA3.1 expressing wt core or the
mutated form C172S. The hepato-
cytes were immunolabeled with
anti-core antibodies, and LDs were
revealed by Red Oil O staining. As
shown in Fig. 6, C172S mutant core
proteins were organized in ring-like
structures around the LDs. At
higher magnification, we noted
FIGURE 2. Palmitoylation of HCV core protein. A,P. pastoris cells expressing
spp (mock) or co-expressing core and spp were induced in medium contain-
ing [
3
H]palmitic acid (50 Ci/mmol), supplemented in some cases with 2-BP (50
"
M). B, Huh7.5 cells were infected with vaccinia virus alone (mock) or with
vaccinia virus expressing HCV core protein in a medium supplemented with
[
3
H]palmitic acid (37
"
Ci/ml) and, when mentioned, with 2-BP (25
"
M). The
cells were disrupted in nonreducing buffer. The samples were resolved on
nonreducing SDS-polyacrylamide gels. The gel was blotted onto a nitrocellu-
lose filter and exposed to a [
3
H] intensifying screen for 7 days. The bands were
revealed using phosphorimaging. The arrow shows the position of the core
protein revealed by Western blotting with anti-core antibodies. Molecular
markers (Bio-Rad) are indicated on the left in kDa.
FIGURE 3. Identification of the palmitoylated cysteine of HCV core by biotin switch assay. Total cell lysates
from yeast P. pastoris expressing spp (mock) or expressing spp and mutant core protein were subjected to the
biotin switch assay as described for Fig. 2. %and #above the lanes indicate hydroxylamine-treated (%)or
untreated (#) protein and neutravidin precipitation pellet (%) or total extract (#). The samples assayed
included core wt, core with C172S or C91L mutations and the double mutant C172S,C91L. Palmitoylated
proteins were analyzed by Western blot using anti-core antibodies.
FIGURE 4. Effect of the Cys
172
mutation on processing of core by spp. A, twenty-four hours after methanol
induction, yeast cells expressing core and the C172S mutant protein with or without (mock) co-expressing spp
were lysed. Protein extracts were separated on SDS-PAGE and revealed by Western blotting using anti-core
antibodies. B, Huh7.5 cells were transfected with pcDNA3.1 plasmid DNA encoding core protein or the C172S
mutant 24 h before protein extraction. Where indicated, hepatocytes were supplemented with 10
"
Mof
(Z-LL)
2
-Ketone. Core proteins were analyzed by Western blot using anti-core antibodies. The arrows on the
right of each panel indicate the unprocessed (gray) and mature (black) forms of the core protein.
Palmitoylation of HCV Core
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that C172S protein was distributed evenly around the LDs with
a smooth ring surface (Fig. 6, Iand J), in 1contrast to the wt
proteins, which showed irregular circle structures with dots
and extra material on the surface of the ring (Fig. 6, Gand H). It
has been described before that core protein triggers ER mem-
brane association to LDs with irregular distribution around the
surface of the organelles (11, 34). Core protein was also
detected with these ER membranes (11, 34). The difference in
pattern distribution between the C172S mutant and the wt pro-
tein may suggest that C172S protein is less present on ER mem-
branes that are closely associated with LDs.
To analyze the distribution of C172S mutant protein to ER
membranes, microsomal membranes were purified from extracts
of Huh7.5 cells transfected with pcDNA3.1 plasmid expressing wt
core or the mutated C172S protein and analyzed by Western blot-
ting using anti-core antibodies. We did not detect any difference
between the amount of wt and C172S protein present in frac-
tions after ER extraction (Fig. 7, lane T). However, during ER
purification, LD fractions are discarded by ultracentrifugation,
possibly with the ER membranes closely associated with LDs.
The ER fractions were further analyzed by density gradient
fractionation. Interestingly, we noted a difference in core asso-
ciation with ER between the C172S and the wt protein. The wt
protein was associated with the ER dense fraction (rough ER) as
well as with the lighter ER membrane fraction (smooth ER).
Core C172S protein was present only with dense ER mem-
branes. It has been shown that ER membranes closely associ-
ated with LDs are free of ribosomes (35). So, according to the
results of our density fractionation experiment, which showed
that the C172S protein has less affinity for smooth ER, we
expected that C172S protein to be absent from the smooth ER
associated with LD.
To investigate whether palmitoylation of Cys
172
may have an
impact on the retention of core in ER/LD-associated mem-
branes, we examined EM micrographs of P. pastoris cells
expressing core. P. pastoris cells have only a few LD/cell, but
these organelles are larger (0.3–0.4, up to 1.6
"
m in diameter)
than what is seen in human cells (10 –100 nm in diameter) (36).
Therefore, it is easier to observe
these organelles and their associa-
tion to ER by immunogold labeling
in yeast. In P. pastoris, wt core was
localized around LD and also on ER
membranes, which appear thicker
than normal (Fig. 8, A,C,D, and F).
However, C172S showed localiza-
tion only around LD and not with
ER membranes (Fig. 8, B,E, and G).
In contrast to wt protein, C172S
proteins were also detected in the
nucleus. Stacks of ER membranes
were easily observed around the LD
of wt core (Fig. 8F). These patterns
were not observed for the C172S
mutant or for cells that were not
expressing core (Fig. 8G); LDs
appeared free of ER membranes.
These results suggest that palmitoy-
lation of core protein at residue Cys
172
helps to recruit ER to the
LDs and/or increases the affinity of core for ER-associated LDs.
Effect of Cys
172
Mutation on LD Accumulation—Earlier stud-
ies have shown that lipid accumulation is significantly greater
in cells expressing core protein (12, 37). The LD induced by
core protein genotype 1a tend to be larger than those present in
naive cells (37). Lipid droplets observed in sections of Huh7.5
cells expressing C172S (Fig. 6) appeared smaller than LD of cells
expressing wt core. To evaluate whether the C172S mutation
affects LD accumulation, the cumulative area of LD in Huh7.5
cell sections was evaluated using Cell profiler software (26). As
expected, the cumulative area of LD was significantly greater
for cells expressing wt core protein than for cells expressing the
plasmid alone (p&0.01) (Fig. 9A). In contrast, sections of cells
expressing the mutant C172S did not reveal any significant
increase in LD area as compared with mock transfected cells.
Mutant C91L showed an accumulation of LD that was similar
to that of wt core.
The analysis of LD accumulation was also performed on
yeast cells. EM micrographs were analyzed for LD area vari-
ations using Cell profiler software. Similar results were
obtained in the P. pastoris system; cumulative areas of LD
were significantly higher in cells expressing wt core and
C91L than in naive cells or those transfected with the mutant
C172S (Fig. 9B). Thus, the C172S mutation clearly affected
the ability of core to induce the accumulation of LD in both
yeast and human cells.
Effect of Cys
172
Mutation on Particle Formation—To deter-
mine the contribution of palmitoylation of core protein to
particle formation, we analyzed the production of nucleo-
capsid-like particles (NLPs) (density, 1.11 g/ml) in yeast
expressing core or its mutated form. P. pastoris expressing
wt core was previously showed to produce NLPs enclosed in
ER membranes that are similar in size and density to HCV
virus particles (20, 38). Protein extracts from P. pastoris co-
expressing spp with core or core mutant were isolated on
sucrose gradients and analyzed by enzyme-linked immu-
nosorbent assay with anti-core antibodies. As observed in
FIGURE 5. Association of HCV core and C172S mutant proteins with DRMs. Huh7.5 cells expressing core or
C172S were lysed, and the aliquots were incubated at 4 °C in the absence (A) or presence (B) of 1% Triton X-100
(Tx-100). The lysates were mixed with Optiprep to a final concentration of 40% iodixanol and then overlaid with
2.9 ml of 30% iodixanol and 400
"
l of TNEi buffer. Following ultracentrifugation, the fractions were collected
from the top of the tube, and the proteins were analyzed by Western blotting using anti-core, anti-caveolin-1
(cav-1), or anti-calnexin (caln) antibodies.
Palmitoylation of HCV Core
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Fig. 10, proteins present in the
fraction at a density of 1.11 g/ml
(20) were drastically reduced by
the C172S mutation as compared
with wt protein. From this results
we concluded that palmitoylation
of the core is necessary for the for-
mation of NLPs in yeast cells.
Effect of Cys
172
Mutation on HCV
Infectivity—To determine the con-
tribution of palmitoylation of core
protein to virus infectivity, we
mutated residue Cys
172
in the con-
text of the J6/JFH1 genome. The
J6/JFH1 construct used (i.e. FL-J6/
JFH1 C19Rluc2AUbi clone) ex-
presses a luciferase protein that is
FIGURE 6. Immunofluorescence analysis of the intracellular distribution of HCV core. Huh7.5 cells were grown on coverslips and transfected with
pcDNA3.1/core (A–C,G, and H) or pcDNA3.1/C172S (D–F,I, and J) using FuGENE 6 transfection reagent. Two days after transfection, the cells were stained with
anti-core antibodies. Lipid droplets were stained with Red Oil O after immunostaining. Bar,5
"
m. Insets and G–J show magnified images. The arrows indicate
ER membranes associated to LD.
FIGURE 7. Isolation and fractionation of ER from Huh7.5 cells expressing HCV core and C172S mutant
proteins. ER membranes from Huh7.5 cells were isolated by ultracentrifugation and then fractionated by
10 –30% discontinuous iodixanol gradients. Lanes 1–18, fractions (0.5 ml) were collected from the top of the
tube and analyzed by Western blotting using an antibody against HCV core protein and calnexin (caln). Lane T
represents the total protein fraction before the Optiprep gradient.
Palmitoylation of HCV Core
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cleaved from the polyprotein to generate a full-length HCV
protein (28). Huh7.5 cells were electroporated with in vitro
transcribed RNA generated from each construct. Luciferase
activity in the cell lysates was measured 4 h post-electropora-
tion as control of transfection efficiency and 72 h post-elec-
troporation as RNA replication efficiency. We observed a
FIGURE 8. Electron micrograph sections of P. pastoris expressing HCV core. Immunogold labeling with anti-core antibodies on thin sections of P. pastoris
cells producing HCV core wt (A,C,D, and F) or C172S (B,E, and G). H, immunostaining of mock transfected cells. N, nucleus; P, peroxisome; M, mitochondria; V,
vacuole: CW, cell wall. Gray,white, and black arrows indicate the core proteins in ER, LD, and nucleus, respectively. Bars, 0.2
"
m.
Palmitoylation of HCV Core
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similar level of transfection for both constructs (data not
shown). Furthermore, the mutation C172S was not signifi-
cantly affected by RNA replication compared with the wild
type J6/JFH1 construct (Fig. 11A). The production of infec-
tious virions was tested at 72 h post-electroporation by inoc-
ulation of naive Huh7.5 cells with filtered cell culture super-
natants. Cell-associated luciferase activity was measured
72 h post-inoculation to quantify infectivity. In contrast to
the robust infectious virus production of wild type J6/JFH1,
the genome containing the C172S mutation in core failed to
produce detectable levels of infectious virus (Fig. 11B). To
determine whether the C172S mutation affected the release
of the virions in the supernatant, we quantified by quantita-
tive reverse transcription-PCR the amount of viral RNA
released from the transfected cells 72 h after electroporation.
As showed in Fig. 11C, the level of viral RNA detected in
J6/JFH1-C172S construct was significantly reduced as com-
pared with the wt. This result indicated that palmitoylation
of core is important for virion assembly or/and for efficient
release of the virion outside the infected cells.
DISCUSSION
For many proteins, the primary function of palmitoylation is
to modify surface hydrophobicity and enhance membrane
affinity, allowing the modified protein to interact with mem-
branes (39). This post-translational modification plays an
important role in protein stability (40), intracellular protein
trafficking, and targeting to membrane microdomains (39).
Palmitoylation is also crucial for assembly and budding of many
viruses (41–44). Unlike other lipid modifications, palmitoyla-
tion is a reversible covalent modification, allowing dynamic
regulation of multiple complex cellular systems. Palmitoylation
occurs at multiple subcellular sites, from the point of synthesis
in the ER, along the secretory pathway, and at the plasma mem-
brane (45). Lipid modifications of a given protein, as well as
palmitoylating enzymes, are conserved from yeast to humans
(17, 46).
In this paper, we present evidence that the HCV core is
modified by palmitoylation in yeast. Importantly, we dem-
onstrate that this post-translational modification is also
occurring in human cells, which provides additional credence
to our findings. Consistent with our results, numerous studies
have proven the reliability of yeast cells for palmitoylation stud-
ies (46– 48). The prediction of core palmitoylation by the algo-
rithm CSS-Palm 2.0 highlighted the fact that Cys
172
is modified
only in the context of the mature core protein after cleavage by
spp and not in the context of the
polyprotein before sp or spp cleav-
age. Accordingly, we observed that
the C172S mutant could be pro-
cessed by spp cleavage as efficiently
as the wt core, suggesting that
Cys
172
palmitoylation does not
influence spp cleavage.
We identified core protein resi-
due Cys
172
as the major palmitoyla-
tion site. Cys
172
is present at the C
terminus of the protein, in close
proximity to the hydrophobic
amino acid sequence that anchors
the protein to the ER membrane (aa
174–191). Interestingly, palmitoy-
lation sites are often found in close
FIGURE 9. Effect of HCV core expression on LD accumulation in Huh7.5
cells (A) and P. pastoris (B). A, after immunostaining of Huh7.5 cells with
anti-core antibodies and LD coloration with Red Oil, the images (35 micro-
graphs/sample) were analyzed for LD content using Cell profiler software
(25). B, EM micrographs of P. pastoris cells expressing spp and co-expressing
spp and HCV core, C172S and C91L were analyzed with Cell profiler software
to determine the area occupied by LD/cell. 20 micrographs for each sample,
representing !300 cells in total, were used for the analysis. The values were
normalized, with mock cells assigned a value of 1. The asterisks denote signif-
icant differences versus mock cells (p&0.05).
FIGURE 10. Effect of Cys
172
mutations in HCV core on particle formation in
yeast. Yeast extracts with similar core protein concentration were loaded on
a 10 –60% (w/w) sucrose gradient. After sedimentation, fractions containing
NLPs at 1.11 g/ml were analyzed by enzyme-linked immunosorbent assay
with anti-core antibodies. The results were normalized on the value of wt
protein content.
FIGURE 11. Effect of Cys
172
mutations on HCV infectivity. A, Huh7.5 cells were transfected in parallel with
RNA transcripts from J6/JFH1 and J6/JFH1-C172S. Replication was assayed by luciferase activity at 72 h post-
electroporation. B, infectious virus production at 72 h post-electroporation was also assayed by luciferase
activity 3 days post-infection. C, detection by quantitative reverse transcription-PCR of the total viral RNA
released 72 h after electroporation of the transcripts transfected in A. The means and S.E. of data from quad-
ruplicate of three different electroporations are shown. The values were normalized on the J6/JFH1 virus data.
The asterisks denote significant differences versus J6/JFH1 (wt) infected cells (p&0.05).
Palmitoylation of HCV Core
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proximity to hydrophobic amino acid stretches of a transmem-
brane domain (TMD) (18, 48). Palmitoylation at these sites is
predicted to increase the effective hydrophobic strength of the
TMD and/or modify orientation of the TMD with respect to the
plane of the lipid bilayer (18). Because the TMD of core is
cleaved by spp to generate a protein of 177 aa (6), the length of
the hydrophobic tail is reduced by 4 amino acids and is likely to
be less efficient at stabilizing attachment of the protein to ER
membranes. Palmitoylation of residue Cys
172
probably helps
maintain the core in close association with ER membranes.
Other structures within core are involved in core-ER stability;
the protein domain from aa 116 to 167 with predicted
amphipathic
!
-helix structures is known to contribute to inter-
action of core with the ER after maturation of the protein by spp
(8, 49). It is likely that this domain and palmitoylation of Cys
172
ensure a stable interaction between core and the ER.
The Cys
172
residue of core protein is well conserved among
different HCV genotypes. Only strain JFH1 has an Phe at
residue 172. For certain proteins, the substitution of the pal-
mitoylated Cys by a hydrophobic residue could compensate
for the absence of the lipid moiety (18, 50). We have some
evidence that it is also the case for the core of JFH1, and this is
under investigation.
3
Palmitoylation has been proposed to enhance the affinity of pro-
teins, both soluble and transmembrane, to cholesterol-rich raft-
like domains (51). Recently core protein was shown to be associ-
ated with DRMs or rafts (14, 30). The DRMs where core
accumulates have properties that distinguish them from classi-
cal plasmalemmal lipid rafts (14). However, our results show
that palmitoylation of core was not involved in DRM targeting
because the C172S mutant was also present in the DRM frac-
tion after Triton solubilization.
We found that, as for the wt core protein, the mutant core
protein C172S co-localized to the surface of LD. The C172S
protein produced in Huh7.5 showed a uniform pattern of dis-
tribution around the LDs as compared with the wt protein,
which exhibited irregular ring structures. It was suggested that
core protein was present on the surface of LDs as well as in the
ER in close proximity to the LD (11, 34); the outside surface of
the large ring structures corresponds to stacks of ER mem-
branes (11). Because the C172S mutant showed sharper and
uniform rings around the LD, we deduced that C172S was
absent from the ER and/or was deficient in recruiting ER mem-
branes around LD structures. This result was confirmed by EM
analysis of yeast expressing core; in yeast, mutation C172S
affected the accumulation of core at ER membranes. Also, in
these cells, we did not observe the stacking of ER membranes
around LD that was easily identifiable in cells expressing wt
core protein. We also found accumulation of C172S core, but
not wt, in the nucleus. Consistently, accumulation of core in the
nucleus was previously described in cells expressing mutant
core proteins that have low affinity for ER membranes (9, 33,
52). In density gradient centrifugation of the ER-purified fraction
from hepatocytes, we observed that affinity for smooth ER was
affected by the C172S mutation. ER membranes associated with
LD are smooth ER (35); therefore, C172S mutant has a reduced
capacity to associate with ER membranes surrounding LD.
Core protein has been shown to trigger lipid accumulation in
cell culture as well as in live animals (53). HCV core protein may
interfere with lipid metabolism on three levels: impaired secre-
tion, increased neosynthesis, and impaired degradation (53).
Triglyceride accumulation is critical for HCV infection (54).
The accumulation of lipid induced by core is certainly helpful
in the establishment of viral infection. We found that the C172S
mutant protein was less efficient in inducing LD accumulation
in Huh7.5 than the wt protein; this impairment of lipid accu-
mulation could have a direct impact on HCV infectivity.
We showed that palmitoylation of Cys
172
was important for
self-assembly of core in yeast cells. In vitro models of particle
assembly do not require the C-terminal part of the protein (19,
55); however, assembly in yeast cells required the hydrophobic
domain including Cys
172
(19). Recently it was found that lipid
modification, in the form of myristoylation, was essential for
multimerization of human immunodeficiency virus Gag in
mammalian cells (56), despite an in vitro model for human
immunodeficiency virus assembly that does not require myris-
tolated Gag protein. Membrane interactions could enhance
protein-protein interactions by nucleation of the protein to a
specific location that triggers the self-assembly process. In our
yeast system, it is likely that this multimerization occurs at the
ER membrane rather than at the LD because C172S core pro-
tein present on LD but absent from the ER cannot trigger par-
ticle formation.
Oligomerization of HCV core and virion assembly has been
suggested to take place at the ER membranes that are closely
associated with LD (11, 34). The association of core protein to
the LD is an essential step in the production of infectious viral
particles (57). According to our results, the association of core
to LD is not sufficient per se for encapsidation. The movement
of core toward ER associated with LD is also a critical parameter
for the efficiency of virus production. One possible function of
the palmitoylation of core is the retention of core in smooth ER
membranes or the redistribution of core proteins from LD to
ER membranes. Because we observed a decrease in ER mem-
brane stacking around LD in C172S-expressing cells, the reten-
tion of ER in LD is possibly improved upon palmitoylation
of core. Impairment of core palmitoylation also affects the
amount of LD accumulation. All of these factors can contribute
to reducing the formation and secretion of HCV particles in
cells infected with the mutated virus C172S.
In conclusion, we showed for the first time that HCV core
protein is modified by palmitoylation at residue Cys
172
,
which is in proximity to ER membranes. Palmitoylation of
Cys
172
controls the association of core to ER membranes
following spp processing, and this association is essential for
virion production.
Acknowledgment—We thank Helen Rothnie for editing of the
manuscript.
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Palmitoylation of HCV Core
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