JOURNAL OF VIROLOGY, Aug. 2008, p. 7964–7976
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 16
Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core
Protein Is Critical for the Production of Infectious Virus Particles?
Takahiro Masaki,1Ryosuke Suzuki,1Kyoko Murakami,1Hideki Aizaki,1Koji Ishii,1Asako Murayama,1
Tomoko Date,1Yoshiharu Matsuura,2Tatsuo Miyamura,1Takaji Wakita,1and Tetsuro Suzuki1*
Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan,1and Department of
Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita-shi, Osaka 565-0871, Japan2
Received 17 April 2008/Accepted 22 May 2008
Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions
in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved
in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus
production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for
HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency
of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine
substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433)
impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of
the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster
with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interac-
tion and virion production, suggesting that phosphorylation of these serine residues is important for virion
production. In addition, we found that the alanine substitutions in the serine cluster suppressed the associ-
ation of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly.
These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by
interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core
Hepatitis C virus (HCV) infection is a major public health
problem and is prevalent in about 200 million people world-
wide (27, 40, 42). Current protocols for treating HCV infection
fail to produce a sustained virological response in as many as
half of treated individuals, and many cases progress to chronic
liver disease, including chronic hepatitis, cirrhosis, and hepa-
tocellular carcinoma (15, 31, 35, 43).
HCV is a positive-strand RNA virus classified in the Hepa-
civirus genus within the Flaviviridae family (55). Its approxi-
mately 9.6-kb genome is translated into a single polypeptide of
about 3,000 amino acids (aa), in which the structural proteins
core, E1, and E2 reside in the N-terminal region. A crucial
function of core protein is assembly of the viral nucleocapsid.
The amino acid sequence of this protein is well conserved
among different HCV strains compared to other HCV pro-
teins. The nonstructural (NS) proteins NS3-NS5B are consid-
ered to assemble into a membrane-associated HCV RNA rep-
licase complex. NS3 possesses the enzymatic activities of serine
protease and RNA helicase, and NS4A serves as a cofactor for
NS3 protease. NS4B plays a role in the remodeling of host cell
membranes, probably to generate the site for the replicase
assembly. NS5B functions as the RNA-dependent RNA poly-
merase. NS5A is known to play an important but undefined
role in viral RNA replication.
NS5A is a phosphoprotein that can be found in basally
phosphorylated (56 kDa) and hyperphosphorylated (58 kDa)
forms (49). Comparative sequence analyses and limited prote-
olysis of recombinant NS5A have demonstrated that NS5A is
composed of three domains (52). Domain I is relatively con-
served among HCV genotypes compared to domains II and
III. Analysis of the crystal structure of the conserved domain I
that immediately follows the membrane-anchoring ?-helix lo-
calized at the N terminus revealed a dimeric structure (53).
The interface between protein molecules is characterized by a
large, basic groove, which has been proposed as a site of RNA
binding. In fact, its RNA binding property has been demon-
strated biochemically (17). Domains II and III of NS5A are far
less understood. Domain II contains a region referred to as the
interferon sensitivity determining region, and this region and
its C-terminal 26 residues have been shown to be essential for
interaction with the interferon-induced, double-stranded
RNA-dependent protein kinase (6–10, 38, 39, 48). Domain III
includes a number of potential phosphoacceptor sites and is
most likely involved in basal phosphorylation. This domain
tolerates insertion of large heterologous sequences such as
green fluorescent protein (GFP) and is not required for func-
tion of NS5A in HCV RNA replication (1, 34). However, a
study with the recently established productive HCV cell cul-
ture system using genotype 2a isolate JFH-1 (28, 56, 58) dem-
onstrated that while insertion of GFP within the NS5A region
does not affect RNA replication, it does produce marked de-
creases in the production of infectious virus particles (41). This
suggests that the C-terminal region of NS5A may affect virus
particle production independent of RNA replication. Re-
* Corresponding author. Mailing address: Department of Virology
II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-
ku, Tokyo 162-8640, Japan. Phone: 81 3 5285 1111. Fax: 81 3 5285
1161. E-mail: email@example.com.
?Published ahead of print on 4 June 2008.
cently, Miyanari et al. reported that the association of core
protein with the NS proteins and replication complexes around
lipid droplets (LDs) is critical for producing infectious viruses
In the present study, we demonstrated that NS5A is a pre-
requisite for HCV particle production via its interaction with
core protein, and we identified serine residues in the C-termi-
nal region of NS5A that play an important role in virion pro-
duction. Substitution of the serine residues with alanine resi-
dues inhibited not only the interaction of NS5A with core
protein but also HCV RNA-core association and led to a
decrease in HCV particle production with no effect on RNA
MATERIALS AND METHODS
DNA construction. Plasmids pJFH1, which contains the full-length JFH-1
cDNA downstream of the T7 RNA promoter sequence, and pSGR-JFH1/Luc, in
which the neomycin resistance gene of pSGR-JFH1 has been replaced by the
firefly luciferase reporter gene, have been previously described (24, 56). To
generate the fluorochrome gene-tagged full-length JFH-1 plasmid, pJFH1/
NS5A-GFP, the region encompassing the RsrII site of NS5A and the BsrGI site
of NS5B was amplified by PCR, the amplification product was cloned into
pGEM-T Easy vector (Promega, Madison, WI), and the resultant plasmid was
designated pGEM-JFH1/RsrII-BsrGI. A GFP reporter gene was amplified by
PCR from pGreen Lantern-1 (Invitrogen, Carlsbad, CA) with primers containing
the XhoI sequence and inserted, after restriction digestion with XhoI, into the
XhoI site of pGEM-JFH1/RsrII-BsrGI. The resulting plasmid was digested by
RsrII and BsrGI and ligated into pJFH1 similarly digested by RsrII and BsrGI to
produce pJFH1/NS5A-GFP. For generation of the fluorochrome gene-tagged
subgenomic reporter plasmid, pJFH1/NS5A-GFP was digested by RsrII and
SnaBI and ligated into pSGR-JFH1/Luc similarly digested by RsrII and SnaBI.
The mutations in the NS5A gene were generated by oligonucleotide-directed
mutagenesis (57). To construct plasmids expressing N-terminally FLAG-tagged
HCV core protein or hemagglutinin (HA)-tagged NS5A, DNA fragments en-
coding core protein or NS5A (wild type or mutants) were generated from the
full-length JFH-1 cDNA by PCR. The core protein coding sequence, together
with a FLAG sequence linked to its N terminus, was cloned into the pCAGGS
vector (37). The coding sequences of NS5A, together with an HA sequence
linked to their N termini, were also cloned into pCAGGS vectors. All PCR
products were confirmed by automated nucleotide sequencing with an ABI Prism
3130 Avant Genetic Analyzer (Applied Biosystems, Tokyo, Japan).
Cells and viruses. The human hepatoma cell line, Huh-7, and JFH1/4-1 cells,
which are Huh-7 cells carrying a subgenomic replicon of JFH-1 (32), were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with minimal essential medium nonessential amino acids (Invitrogen), 100
units/ml of penicillin, 100 ?g/ml of streptomycin, and 10% fetal bovine serum
(FBS) at 37°C in a 5% CO2incubator. Huh/c-p7 cells, which are Huh-7 cells
stably expressing the proteins core to p7 derived from the JFH-1 strain (18), were
incubated in DMEM containing 300 ?g/ml of zeocin (Invitrogen). HCV particles
derived from JFH-1 were produced by transient transfection of Huh-7 cells with
in vitro transcribed RNA, as described previously (56, 58). Recombinant vaccinia
virus strain DIs, which expresses the bacteriophage T7 RNA polymerase under
the control of the vaccinia virus early/late promoter P7.5, was generated and
propagated as previously described (19).
DNA transfection, immunoprecipitation (IP), and immunoblotting. For coex-
pression of FLAG-tagged core protein and HA-tagged NS5A, cells were seeded
onto 35-mm wells of a six-well cell culture plate and cultured overnight. Plasmid
DNAs (2 ?g) were transfected into cells using TransIT-LT1 transfection reagent
(Mirus, Madison, WI). Cells were harvested at 48 h posttransfection, washed
three times with 1 ml of ice-cold phosphate-buffered saline (PBS), and sus-
pended in 0.25 ml lysis buffer (20 mM Tris-HCl [pH 7.4] containing 135 mM
NaCl, 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], and 10% glycerol)
supplemented with 50 mM NaF, 5 mM Na3VO4, 1 ?g/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride (PMSF). Cell lysates were sonicated at 4°C for 5
min, incubated for 30 min at 4°C, and centrifuged at 14,000 ? g for 5 min at 4°C.
After preclearing, the supernatant was immunoprecipitated with 10 ?l of anti-
FLAG M2-agarose beads (Sigma, St. Louis, MO). For expression of the full-
length HCV polyprotein, Huh-7 cells transfected with 10 ?g of in vitro tran-
scribed RNAs by electroporation were resuspended in 20 or 30 ml of culture
medium, and 10-ml aliquots were seeded into 100-mm culture dishes. At 72 h
posttransfection, the cells were incubated in 0.5 ml of lysis buffer (20 mM
Tris-HCl [pH 7.4] containing 135 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, and 10% glycerol) supplemented with 50 mM NaF, 5 mM
Na3VO4, 1 ?g/ml leupeptin, and 1 mM PMSF. After preclearing, the supernatant
was immunoprecipitated with 5 ?g of polyclonal anti-NS5A antibody (34a) or
polyclonal anti-C/EBP? antibody (Santa Cruz Biotechnology, Santa Cruz, CA),
and 20 ?l of protein G-agarose beads (Invitrogen). The immunocomplex was
precipitated with the beads by centrifugation at 800 ? g for 30 s and then was
washed five times with lysis buffer by centrifugation. The proteins binding to the
beads were boiled in 20 ?l of SDS sample buffer and then subjected to SDS–
12.5% polyacrylamide gel electrophoresis (PAGE). The proteins were trans-
ferred onto a polyvinylidene difluoride membrane (Immobilon; Millipore, Bed-
ford, MA) and then reacted with a primary antibody and a secondary horseradish
peroxidase-conjugated antibody. The immunocomplexes were visualized with an
ECL Plus Western Blotting Detection System (GE Healthcare, Buckingham-
shire, United Kingdom) and detected using an LAS-3000 imaging analyzer (Fu-
jifilm, Tokyo, Japan).
In vitro synthesis of HCV RNA and RNA transfection. Plasmid DNAs were
digested with XbaI and treated with mung bean nuclease (New England Biolabs,
Ipswich, MA) to remove the four terminal nucleotides, resulting in the correct 3?
end of the HCV cDNA. Digested DNAs were purified and used as templates for
RNA synthesis. HCV RNA was synthesized in vitro using a MEGAscript T7 kit
(Ambion, Austin, TX). Synthesized RNA was treated with DNase I (Ambion),
followed by acid guanidinium thiocyanate-phenol-chloroform extraction to re-
move any remaining template DNA. Synthesized HCV RNAs were used for
electroporation. Trypsinized Huh-7 cells were washed with Opti-MEM I re-
duced-serum medium (Invitrogen) and resuspended at 3 ? 106cells/ml with
Cytomix buffer (54). RNA was mixed with 400 ?l of cell suspension and trans-
ferred into an electroporation cuvette (Precision Universal Cuvettes; Thermo
Hybaid, Middlesex, United Kingdom). Cells were then pulsed at 260 V and 950
?F using a Gene Pulser II unit (Bio-Rad, Hercules, CA). Transfected cells were
immediately transferred onto six-well culture plates or 100-mm culture dishes.
Luciferase assay. Cells were harvested at different time points posttransfection
of subgenomic reporter replicons and lysed in passive lysis buffer (Promega). The
luciferase activity in cells was determined using a luciferase assay system (Pro-
Quantification of HCV core protein. HCV core protein in transfected cells or
cell culture supernatants was quantified using a highly sensitive enzyme immu-
noassay (Ortho HCV antigen ELISA Kit; Ortho Clinical Diagnostics, Tokyo,
Japan). To determine intracellular core protein amounts, cell lysates were pre-
pared as described previously (41). To determine the efficiency of core protein
release, the ratio of extracellular core protein to total core protein (the sum of
intra- and extracellular core protein amounts) was calculated.
Intra- and extracellular infectivity assay. Culture supernatants were harvested
72 h posttransfection, and virus titers were determined by a 50% tissue culture
infectious dose (TCID50) assay as described previously (28, 46). Virus titration
was performed by seeding naı ¨ve Huh-7 cells in 96-well plates at a density of 1 ?
104cells/well. Samples were serially diluted fivefold in complete growth medium
and used to infect the seeded cells (six wells per dilution). At 72 h after infection,
the inoculated cells were fixed and immunostained with a mouse monoclonal
anti-core protein antibody (2H9) (56), followed by an Alexa Fluor 488-conju-
gated anti-mouse immunoglobulin G (IgG) (Invitrogen). Wells that showed at
least one core protein-expressing cell was counted as positive. Cell-associated
infectivity was determined essentially as described previously (12, 47). Briefly,
cells were extensively washed with PBS, scraped, and centrifuged for 3 min at
120 ? g. Cell pellets were resuspended in 1 ml of DMEM containing 10% FBS
and subjected to four cycles of freezing and thawing using dry ice and a 37°C
water bath. Samples were then centrifuged at 2,400 ? g for 10 min at 4°C to
remove cell debris, and cell-associated infectivity was determined by TCID50
Expression of HCV proteins using vaccinia viruses, metabolic labeling of cells,
and radioimmunoprecipitation analysis. Metabolic labeling of cells and radio-
immunoprecipitation analysis were performed as described by Huang et al. (17)
with some modifications. A total of 4 ? 105Huh-7 cells were seeded onto each
well of six-well cell culture plates and cultured overnight. A 2-?g amount of
subgenomic replicon DNAs carrying defined NS5A mutations was transfected
into cells using TransIT-LT1 transfection reagent, and at 12 h posttransfection
the cells were then infected at a multiplicity of infection of 10 with recombinant
vaccinia viruses expressing the T7 RNA polymerase. After 40 h of transfec-
tion, cells were incubated in methionine- and cysteine-deficient DMEM (In-
vitrogen) or phosphate-deficient DMEM (Invitrogen) for 2 h and labeled for
6 h with [35S]methionine and [35S]cysteine (200 ?Ci/well; GE Healthcare) or
VOL. 82, 2008 ROLE OF HCV NS5A-CORE INTERACTION IN VIRION PRODUCTION7965
[32P]orthophosphate (250 ?Ci/well; GE Healthcare). The cells were then washed
twice with cold PBS and lysed with SDS lysis buffer (50 mM Tris-HCl [pH 7.6],
0.5% SDS, 1 mM EDTA, 20 ?g/ml of PMSF). The cell lysates were passed
through a 27-gauge needle several times to shear cellular DNA. After a 10-min
incubation at 75°C, the lysates were clarified by centrifugation and diluted five-
fold with HNAET buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 0.67% bovine
serum albumin, 1 mM EDTA, 0.33% Triton X-100). After preclearing by incu-
bation with 20 ?l of protein G-agarose beads for 1 h at 4°C, the supernatant was
incubated with 2 ?g of rabbit polyclonal anti-NS5A antibody overnight at 4°C. A
20-?l aliquot of protein G agarose beads was further added and incubated for 2 h
at 4°C. The cell pellets were washed three times with 0.5 ml of HNAETS buffer
(HNAET containing 0.5% SDS), followed by washing once with 0.5 ml of HNE
buffer (50 mM HEPES [pH 7.5], 150 mM NaCl and 1 mM EDTA). After
treatment with or without ? protein phosphatase (New England Biolabs), the cell
pellets were suspended in 20 ?l of SDS sample buffer and boiled for 10 min. The
proteins were resolved on 10% SDS-polyacrylamide gels and analyzed by auto-
Subcellular fractionation analysis. All steps were carried out at 4°C in the
presence of a protease inhibitor cocktail (Complete; Roche, Mannheim, Ger-
many) as described previously (20), with some modifications. Cells were sus-
pended in four cell volumes of homogenization buffer (50 mM NaCl, 10 mM
triethylamine [pH 7.4], 1 mM EDTA), snap frozen in liquid nitrogen, stored at
?80°C, and thawed in a water bath at room temperature. Supernatants (0.4 ml)
were layered on linear 10-ml iodixanol gradients from 2.5 to 25% and centrifuged
at 37,000 rpm for 3.5 h in an SW41 rotor (Beckman, Fullerton, CA), followed by
collection of 0.8-ml fractions from the top. Each fraction was concentrated by
Centricon YM30 (Millipore), separated by SDS-PAGE, and immunoblotted with
a rabbit polyclonal anti-calnexin antibody (Stressgen Biotechnologies, Victoria,
Canada), a mouse monoclonal anti-adipose differentiation-related protein
(ADRP) antibody (Progen Biotechnik, Heidelberg, Germany), or a rabbit poly-
clonal anti-NS5A antibody. The core protein amount in each fraction was also
determined by enzyme-linked immunosorbent assay (ELISA).
IP-RT-PCR. The process of cell lysis to RNA purification was carried out
essentially as described by Johnson et al. (21) with some modifications. A total of
3 ? 106Huh-7 cells were transfected with 10 ?g of in vitro transcribed HCV
RNAs and resuspended in 20 or 30 ml of culture medium, after which 10-ml
aliquots were seeded into 100-mm culture dishes. At 72 h posttransfection, the
cells were scraped and incubated in 500 ?l of hypotonic buffer (10 mM HEPES
[pH 7.6], 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF) per dish. The cells were
passed through a 20-gauge needle several times, lysed with Nonidet P-40 at a
final concentration of 1%, and incubated on ice for an additional 10 min. After
centrifugation at 4,000 ? g at 4°C for 15 min, glycerol was added to the super-
natants at a final concentration of 5%. The cell lysates were incubated with 20 ?l
of protein G-agarose beads for 30 min at room temperature. After the cell lysates
were removed from protein G-agarose beads, 5 ?g of mouse monoclonal anti-
core protein antibody or normal mouse IgG (Sigma) as a negative control was
added, and samples were incubated for an additional 1 h at room temperature.
A 20-?l aliquot of protein G-agarose beads per sample was added to the cell
lysates and incubated for 1 h. After incubation, the beads were washed three
times with wash buffer (10 mM Tris-HCl [pH 7.6], 100 mM KCl, 5 mM MgCl2,
and 1 mM dithiothreitol) and eluted in 100 ?l of elution buffer (50 mM Tris-HCl
[pH 8.0], 1% SDS, and 10 mM EDTA) at 65°C for 10 min. After treatment with
100 ?g of proteinase K at 37°C for 30 min, the RNAs in immunocomplexes were
isolated by acid guanidinium thiocyanate-phenol-chloroform extraction. Reverse
transcriptase PCR (RT-PCR) was carried out using random hexamer and Su-
perscript II RT (Invitrogen), followed by nested PCR with LA Taq DNA poly-
merase (TaKaRa, Shiga, Japan) and primer sets amplifying the fragments of
nucleotides (nt) 129 to 2367 and nt 7267 to 9463 of the JFH-1 genome. To
amplify the fragment of nt 129 to 2367, the sense primer 5?-CTGTGAGGAAC
TACTGTCTT-3? and the antisense primer 5?-TCCACGATGTTCTGGTGAA
G-3? were used for first-round PCR; the sense primer 5?-CGGGAGAGCCAT
AGTGG-3? and the antisense primer 5?-CATTCCGTGGTAGAGTGCA-3?
were used for second-round PCR. To amplify the fragment of nt 7267 to 9463,
the sense primer 5?-GTCCAGGGTGCCCGTTCTGGACT-3? and the antisense
primer 5?-GCGGCTCACGGACCTTTCAC-3? were used for first-round PCR;
the sense primer 5?-CACCGTTGCTGGTTGTGCT-3? and the antisense primer
5?-GTGTACCTAGTGTGTGCCGCTCTA-3? were used for second-round PCR.
Indirect immunofluorescence analysis. Cells incubated for 3 days after trans-
fection with JFH-1 RNAs were seeded in an eight-well chamber slide (BD
Biosciences, San Jose, CA) and cultured overnight. The adherent cells were
washed twice with PBS and fixed with 4% paraformaldehyde at room tempera-
ture. After a washing step with PBS, the cells were permeabilized with PBS
containing 0.3% Triton X-100 and 2% FBS for 1 h at room temperature and
stained with a rabbit polyclonal anti-NS5A antibody and a mouse monoclonal
anti-core protein antibody. The fluorescent secondary antibodies were Alexa
Fluor 488- or Alexa Fluor 555-conjugated anti-rabbit or anti-mouse IgG anti-
bodies (Invitrogen). Analyses of JFH-1 were performed on a Zeiss confocal laser
scanning microscope LSM 510 (Carl Zeiss, Oberkochen, Germany).
Mutations of serine residues at the NS5A C terminus impair
basal phosphorylation but have little effect on viral RNA rep-
lication. As demonstrated in a previous study, insertion of GFP
into the NS5A C terminus does not significantly affect viral
RNA replication but reduces the generation of infectious HCV
particles (41). The C-terminal region of NS5A contains highly
conserved serine residues that are involved in basal phosphor-
ylation (1, 23, 49). To examine the involvement of the serine
clusters (cluster 3-A [CL3A] and cluster 3-B [CL3B]) in the
C-terminal region of NS5A in HCV particle production, we
created mutated HCV genomes as well as subgenomic repli-
cons carrying alanine substitutions for the conserved serine
residues at aa 2384, 2388, 2390, and 2391 (residues are num-
bered according to the positions within the original JFH-1
polyprotein) (CL3A/SA); at aa 2428, 2430, and 2433 (CL3B/
SA); or an in-frame deletion spanning aa 2384 to 2433 (?2384–
2433) (Fig. 1). A construct with an in-frame insertion of GFP
(NS5A-GFP) was also generated as described previously for
the Con1 isolate (34).
First, we analyzed the effects of the NS5A mutations on
HCV RNA replication using a transient RNA replication assay
using subgenomic luciferase reporter replicons (Fig. 2A) and
found that the serine-to-alanine substitutions (CL3A/SA and
CL3B/SA) did not affect viral RNA replication. NS5A-GFP
and ?2384–2433 slightly reduced RNA replication, indicating
that the mutations of the NS5A C terminus tested in this study
do not critically affect RNA replication, which is consistent
with previous reports (1, 34, 51).
Next, the phosphorylation status of the mutated NS5A was
analyzed as described in Materials and Methods (Fig. 2B).
NS5A was isolated from radiolabeled cells by IP and analyzed
either directly by SDS-PAGE or after treatment with ? protein
phosphatase. Analysis of
that the CL3A/SA, CL3B/SA, and ?2384–2433 mutations re-
sulted in marked reduction of basal phosphorylation (Fig. 2B,
compare lane 1 with lanes 3, 5, and 7 in the top panel). All
32P-labeled NS5A proteins were sensitive to treatment with
phosphatase (lanes 2, 4, 6, and 8). The possibility that loss of
signal after dephosphorylation was due to contaminating pro-
teases present in the phosphatase preparations can be ruled
out because no degradation of the35S-labeled proteins was
observed (Fig. 2B, bottom panel). These results suggest that
mutations in the C-terminal serine cluster of NS5A impair
basal phosphorylation but have no significant effect on viral
Effect of mutations introduced into the NS5A C terminus on
the production of infectious HCV particles. To analyze HCV
particle production from cells transfected with the in vitro
transcribed viral genomic RNAs, we harvested supernatants
and cells at 4, 24, 48, 72, and 96 h posttransfection and mea-
sured the amounts of core protein. As shown in Fig. 3A, com-
parable amounts of core proteins were detected in all trans-
fected cells 4 h after transfection, reflecting unchanged
32P-radiolabeled proteins revealed
7966MASAKI ET AL. J. VIROL.
transfection efficiencies, and the kinetics of intracellular core
protein levels was similar among transfectants. By contrast,
core protein released from cells transfected either with the
mutated genome of CL3B/SA, ?2384–2433, or NS5A-GFP was
more than 10-fold lower than that for the wild-type JFH-1 or
CL3A/SA (Fig. 3B). Figure 3C shows the efficiency of core
protein release from each transfectant, which is expressed as a
percentage of the extracellular core protein level relative to the
amount of total core protein (the sum of intra- and extracel-
lular core protein). Core protein release efficiency with the
wild type and CL3A/SA was 2 to 13% at 48 to 96 h after
transfection, while only 1% or less of core protein was released
in the cases of CL3B/SA, ?2384–2433, and NS5A-GFP strains.
To further investigate production and release of infectious
virus particles, naı ¨ve Huh-7 cells were infected with culture
supernatants of cells harvested 72 h posttransfection, and in-
fectious virus titers were determined by TCID50assay at 72 h
after infection. Figure 3D shows that release of infectious virus
particles from cells transfected with the genome of CL3B/SA
or ?2384–2433 mutants was markedly reduced (about 10,000-
fold) compared to that from wild-type- or CL3A/SA-trans-
fected cells (white bars). To examine whether such a decrease
in infectious HCV in the culture supernatants was attributable
to defective virion assembly or impaired release of virions, we
determined cell-associated infectivity (Fig. 3D). Production of
intracellular infectious virions in CL3B/SA- and ?2384-2433-
transfected cells was strongly impaired in comparison with that
in wild-type-transfected (?1,000-fold) and CL3A/SA-trans-
fected (?100-fold) cells. Thus, the results suggest a potential
role for the serine cluster at aa 2428, 2430, and 2433 of NS5A
in assembly of infectious HCV particles. Among the NS5A
mutations tested, CL3B/SA is of particular interest because
this mutation leads to a marked reduction in HCV production
with no impact on viral RNA replication.
Serine residues at aa 2428, 2430, and 2433 are important for
the interaction between NS5A and core protein. Miyanari et al.
reported that the association of core protein with NS proteins
is critical for infectious HCV production and that mutations of
the core protein and NS5A that cause these proteins to fail to
associate with each other impair the production of infectious
virus (33). Based on these observations and the findings noted
above, we hypothesize that NS5A plays a key role in recruiting
viral RNA, which is synthesized at the viral replication com-
plex, to nucleocapsid formation via interaction between the
NS5A C-terminal region and the core protein. To prove this,
we analyzed the interaction of NS5A with the core protein by
coimmunoprecipitation experiments. HA-tagged NS5A con-
structs carrying defined mutations were generated (Fig. 1) and
coexpressed with the FLAG-tagged core protein in Huh-7
cells. As shown in Fig. 4A, coimmunoprecipitation of NS5A
FIG. 1. Structures of HCV constructs used in this study. Schematic diagram of the NS5A structure according to Tellinghuisen et al. (52) is
shown in the top panel. The three domains are indicated by white boxes and are separated by trypsin-sensitive regions with presumably low
structural complexity (low-complexity sequence [LCS]). The numbers indicate amino acid residues within the original JFH-1 polyprotein. The
names listed on the left represent full-length HCV constructs, subgenomic reporter replicons, or N-terminally HA-tagged NS5A constructs used
in this study. NS5A-GFP carries a GFP insertion between aa 2394 and 2395 as indicated by a shaded box. CL3A/SA and CL3B/SA carry several
serine-to-alanine substitutions in the NS5A C terminus constructed as described previously (1). HCV constructs from S2428A to S2430/2433A carry
single or double serine-to-alanine substitutions generated by modification of the CL3B/SA construct. The ?2384–2433 mutant possesses an
in-frame deletion in the C-terminal region of NS5A. Amino acid substitutions are marked in bold and underlined. N and C represent N terminus
and C terminus, respectively.
VOL. 82, 2008 ROLE OF HCV NS5A-CORE INTERACTION IN VIRION PRODUCTION7967
with the core protein was observed in cells expressing the
wild-type NS5A and the CL3A/SA-mutated NS5A, but the
amount of immunoprecipitated NS5A in the CL3A/SA-ex-
pressing cells was slightly lower than that in the wild-type-
expressing cells. In contrast, the CL3B/SA- or the ?2384-2433-
mutated NS5A coimmunoprecipitated with the core protein
only slightly or not at all.
We further examined the interaction of NS5A with core
protein in cells expressing HCV genomes. At 72 h posttrans-
fection with the wild type or CL3B/SA, cells were harvested
and immunoprecipitated with an anti-NS5A antibody or an
anti-C/EBP? antibody as a negative control, followed by
immunoblotting. Under these experimental conditions, the
amount of extracellular core protein released from cells trans-
fected with the CL3B/SA genome was about 10-fold lower than
that for the wild type, although comparable amounts of intra-
cellular core protein were observed in both transfectants (Fig.
4B, left panels). As shown in the right panels of Fig. 4B, the
core protein was specifically coimmunoprecipitated with NS5A
in cells expressing the wild-type JFH-1 genome but not with
the mutated NS5A in cells expressing the CL3B/SA genome.
These results demonstrate that NS5A interacts with the core
protein in cells producing infectious particles and that serine
residues at aa 2428, 2430, and 2433 are important to the suc-
cess of this interaction.
Two serine residues among aa 2428, 2430, and 2433 are
responsible for regulating the interaction of NS5A with the
core protein as well as HCV particle production. To further
determine the critical residues in the C-terminal serine cluster
of NS5A responsible for HCV particle production, we replaced
one or two serine residues in the region with alanine (Fig. 1)
and investigated which serine-to-alanine substitution influ-
enced HCV particle production. Core protein levels in cells
transfected with any construct were comparable over 4 days
after transfection, indicating similar efficiencies of transfection
and RNA replication from each construct (data not shown). As
shown in Fig. 5A, we observed a slight delay in the kinetics of
core protein release from cells transfected with the single-
substitution genomes, S2428A, S2430A, and S2433A, up to 48
or 72 h posttransfection. However, core protein release from
these cells reached comparable levels to that for the wild type
at 96 h after transfection. In the cases of the double-substitu-
tion mutants (Fig. 5B), core protein release from cells trans-
fected with the double-substitution genomes was markedly re-
duced, with 10- to 30-fold decreases compared to that for wild
type observed. The kinetics of core protein release were similar
to that for CL3B/SA.
Interaction of NS5A carrying single or double serine-to-
alanine substitutions with the core protein was investigated by
coimmunoprecipitation analysis using HA-tagged NS5A con-
structs. NS5A mutants carrying a single substitution were co-
immunoprecipitated with the core protein (Fig. 5C), while
none of the double-substitution NS5A mutants or the triple-
substitution mutant, CL3B/SA, coimmunoprecipitated with the
core protein (Fig. 5D). These results suggest that at least two
serine residues in the C-terminal serine cluster of NS5A (aa
2428, 2430, and 2433) are necessary for the interaction be-
tween NS5A and the core protein as well as for regulation of
HCV particle production and that there is positive correlation
between their interaction and the amount of core protein re-
Glutamic acid partially substitutes for serine phosphoryla-
tion in the interaction of NS5A with the core protein and virus
production. A consequence of phosphorylation is the addition of
negative charge to a protein. In some cases, phosphoserine can be
mimicked by glutamic or aspartic acid (14). To determine
whether the introduction of negative charges into aa 2428, 2430,
and 2433 instead of phosphoserines positively regulates the inter-
action of NS5A with the core protein and virus production, we
replaced the serine residues with glutamic acid residues and con-
structed the CL3B/SE and S2428/2430E mutants (Fig. 6A). Cells
transfected with the double-glutamic acid substitution, S2428/
2430E, exhibited similar kinetics to the wild-type-transfected cells
and released ?22-fold more core protein than S2428/2430A-
transfected cells by 96 h posttransfection (Fig. 6B). In contrast,
FIG. 2. Mutations at the C terminus of NS5A impair basal phos-
phorylation and have only a minor impact on RNA replication.
(A) Replication of given mutants in transfected Huh-7 cells as deter-
mined by luciferase reporter assays performed at 24, 48, and 72 h
posttransfection (white, gray, and black bars, respectively). Values
given were normalized for transfection efficiency using the luciferase
activity determined 4 h after transfection, which was set to 1. Mean
values of quadruplicate measurements and the standard deviations are
given. (B) Phosphorylation analysis of NS5A using the vaccinia virus
T7 hybrid system. NS3-to-NS5B polyprotein fragments carrying the
mutations specified above the lanes were transfected into Huh-7 cells,
and proteins were radiolabeled with [32P]orthophosphate or [35S]me-
thionine and [35S]cysteine. NS5A proteins were isolated by IP and
separated by SDS-PAGE (10% polyacrylamide). Mock-transfected
cells served as a negative control (lanes 9 and 10). Half of the samples
were treated with ? protein phosphatase (?-PPase) (?) whereas the
other half was mock treated (?) prior to SDS-PAGE. Arrows and
asterisks indicate hyperphosphorylated and basally phosphorylated
7968MASAKI ET AL. J. VIROL.
the transfectant with the triple glutamic acid substitution, CL3B/
SE, showed similar trends to that of CL3B/SA. In the coimmu-
noprecipitation experiments with FLAG-tagged core protein and
HA-tagged NS5A constructs (Fig. 6C), S2428/2430E, but not
S2428/2430A, restored the ability of NS5A to interact with the
core protein up to a similar level to that of wild type. As expected,
neither CL3B/SE nor CL3B/SA coimmunoprecipitated with the
core protein. Taken together, these results indicate that negative
charges at aa 2428 and 2430 preserve the ability of NS5A to
interact with the core protein and positively regulate virus pro-
duction. However, the data of the CL3B/SE mutant indicate that
either the interaction of NS5A with the core protein or virus
Subcellular localization of NS5A and core protein in Huh-7
cells expressing HCV genomes. The coimmunoprecipitation
experiments described above indicate that the wild-type NS5A
but not the CL3B/SA mutant interacts with the core protein.
To evaluate the NS5A-core protein interaction in intact cells,
we examined the subcellular localization of NS5A with the core
protein by immunofluorescence analysis. NS5A colocalized with
the core protein in cells transfected with the JFH-1 wild type (Fig.
7A), whereas their colocalization was rarely observed in cells
transfected with the CL3B/SA RNA (Fig. 7B).
To further analyze the subcellular compartments for the
localization of NS5A and core protein in cytoplasmic mem-
brane structures, including the endoplasmic reticulum (ER)
and LDs, we performed subcellular fractionation studies as
FIG. 3. Effect of mutations introduced into the NS5A C terminus on the production of infectious HCV particles. (A) Intracellular levels of core
protein measured at various time points after transfection. A total of 3 ? 106Huh-7 cells were transfected with 10 ?g of in vitro-transcribed HCV
RNAs specified in the inset and resuspended in 10 ml of culture medium, after which 2-ml aliquots were seeded into each well of a six-well culture
plate. The cells were harvested at different time points between 4 h and 96 h posttransfection, and then 500 ?l of cell lysate per well was prepared.
After centrifugation, supernatants were processed for a core protein-specific ELISA. (B) Release of core protein from cells transfected with the
HCV genomes specified in the inset. Cell culture supernatants harvested from cells given in panel A were analyzed by a core protein ELISA.
(C) Efficiency of core protein release from cells transfected with the HCV genomes specified in the inset. The percent core protein release (vertical
axis) indicates the percentage of released core protein in relation to total core protein (the sum of intra- and extracellular core protein) calculated
for each time point. (D) Infectivity of virus particles contained in supernatants and cells after transfection with mutants specified below the graph.
Culture supernatants and cells were harvested 72 h posttransfection, and extracellular (white bars) and intracellular infectivity (gray bars) levels
were determined by TCID50assay. The gray line and arrowhead represent the detection limit of the limiting dilution assay. Mean values and
standard deviations for at least triplicates are shown in all panels.
VOL. 82, 2008 ROLE OF HCV NS5A-CORE INTERACTION IN VIRION PRODUCTION 7969
described in Materials and Methods. The iodixanol gradient
was collected from the top to the bottom into 12 fractions
(fractions 1 to 12). As shown in Fig. 7C, an ER marker, cal-
nexin, was found in fractions 7 to 12 and was localized primar-
ily in fractions 11 and 12. In contrast, ADRP, a cellular marker
for LDs, was mainly observed in fractions 4 to 7. These two
markers were equally distributed among cells analyzed (data
not shown). The distribution of the wild-type NS5A was found
in fractions 4 to 7, which was parallel to the fractionation
profile of ADRP. The CL3B/SA-mutated NS5A was more
broadly distributed and was also observed in heavier fractions
than the wild-type NS5A, which was analogous to distribution
of NS5A expressed in JFH1/4-1 cells bearing subgenomic rep-
licons. The core protein in cells expressing the JFH-1 wild type,
the CL3B/SA mutant, and in Huh/c-p7 cells that express JFH-1
structural proteins was distributed in a similar fashion, indicat-
ing that the distribution of core protein is not affected by NS5A
mutation. The fractionation profile of the core protein, with a
peak in fraction 4 or 5, was similar to that of the wild-type
NS5A or ADRP but not to that of the CL3B/SA-mutated
NS5A or calnexin, suggesting that core protein interacts with
the wild-type NS5A in LD fractions, which is consistent with
previous reports (33, 44, 45).
NS5A-core protein interaction is important for association
of the core protein with the viral genomic RNA. To further
address our hypothesis regarding involvement of NS5A in re-
cruiting viral RNA to nucleocapsid formation, we analyzed the
association of the core protein with HCV RNA in wild-type- or
CL3B/SA-expressing cells by IP-RT-PCR (Fig. 8). Both cell
lysates were immunoprecipitated with an anti-core protein an-
tibody or a negative control, mouse IgG. Total RNA prepared
from each immunoprecipitate was subjected to RT-PCR in
order to detect HCV RNA. The amounts of immunoprecipi-
tated core protein (Fig. 8, lower panel) as well as the expres-
sion of HCV RNA (Fig. 8, upper panels, Input) were compa-
rable in both cells. In cells expressing the wild-type JFH-1
genome, the viral RNAs covering the 5? terminal 2.2-kb as well
as the 3? terminal 2.2-kb regions were detected in immunopre-
FIG. 4. aa 2428, 2430, and 2433 are essential for the interaction between NS5A and the core protein. (A) Effect of mutations at the NS5A C
terminus on the interaction of NS5A with the core protein. N-terminally FLAG-tagged core protein and N-terminally HA-tagged NS5A carrying
defined mutations were coexpressed in Huh-7 cells and immunoprecipitated with anti-FLAG antibody. The resulting precipitates were examined
by immunoblotting using anti-HA or FLAG antibody. One-tenth of the cell lysates used in IP is shown as the 10% input. (B) Interaction between
NS5A and the core protein in HCV-replicating cells. Huh-7 cells were lysed 72 h after transfection of the in vitro transcript of the HCV genome
(wild type or CL3B/SA) and were immunoprecipitated with anti-NS5A antibody or anti-C/EBP? antibody as a negative control. The resulting
precipitates were examined by immunoblotting using anti-core protein, NS5A, or C/EBP? antibody. One-tenth of cell lysates used in IP was
immunoblotted with anti-core protein antibody (10% input). Cell culture supernatants harvested from transfected cells were analyzed by a core
protein ELISA in parallel. IB, immunoblotting.
7970 MASAKI ET AL.J. VIROL.
cipitates obtained with the anti-core protein antibody but not
with the mouse IgG. In contrast, in cells expressing the
CL3B/SA genome, HCV RNA was not detected in the immu-
noprecipitates with either antibody. These results demonstrate
that HCV RNA associates with the core protein in cells where
NS5A interacts with core protein (JFH-1 wild type) but not in
cells where their interaction is impaired (CL3B/SA).
In the present study, we demonstrated the involvement of
NS5A in the production of HCV particles via the interaction of
NS5A with the core protein and identified its C-terminal serine
cluster 3-B (aa 2428, 2430, and 2433), which is implicated in
basal phosphorylation, as a key element for the interaction of
NS5A with the core protein and for infectious virus produc-
tion. Serine-to-alanine substitutions at the cluster, which have
no impact on viral RNA replication, inhibit the interaction
between NS5A and the core protein, thereby indicating that
there is a connection between NS5A-core protein association
and virus production. Finally, CL3B mutation leads to impair-
ment of the association of the core protein with HCV RNA
and, therefore, possibly RNA encapsidation.
Several reports have indicated that viral NS proteins are
involved in the virion assembly of Flaviviridae viruses (25, 29,
30, 33). For instance, mutations in yellow fever virus NS2A
block production of infectious virus, and this perturbation can
be released by a suppressor mutation in NS3 (25), while the
hydrophobic residues of Kunjin virus NS2A required for virus
assembly have been mapped (26). Miyanari et al. have shown
that HCV core protein recruits NS proteins to the LD-associ-
ated membranes and that the NS proteins around the LDs
participate in the assembly of infectious viral particles (33).
Furthermore, during preparation of the current article, two
studies regarding participation of NS5A in the assembly of
HCV particles were published. Appel et al. have demonstrated
the essential role of domain III of NS5A in the formation of
infectious particles, and deletions in this domain that disrupt
colocalization of NS5A and the core protein abrogate virion
production (2). Tellinghuisen et al. identified a serine residue
in domain III as a key determinant for viral particle production
FIG. 5. Determination of critical amino acids responsible for virus production and the interaction of NS5A with the core protein. (A and B)
Effect of single or double serine-to-alanine substitutions on virus production. After transfection of in vitro transcripts of the HCV genomes
specified in the inset into Huh-7 cells, the cells and culture supernatants were harvested at the time points given, and the amounts of the core
protein were determined by core protein-specific ELISA. Percent core protein release (vertical axis) indicates the percentage of released core
protein in relation to total core protein (the sum of intra- and extracellular core protein) calculated for each time point. Mean values and standard
deviations for at least triplicate experiments are shown. (C and D) Effect of single or double serine-to-alanine substitutions on the interaction
between NS5A and the core protein. N-terminally FLAG-tagged core protein and N-terminally HA-tagged NS5A carrying defined mutations were
coexpressed in Huh-7 cells and immunoprecipitated with anti-FLAG antibody. The resulting precipitates were examined by immunoblotting using
anti-HA or FLAG antibody. One-tenth of the cell lysates used in IP is shown as the 10% input. IB, immunoblotting.
VOL. 82, 2008 ROLE OF HCV NS5A-CORE INTERACTION IN VIRION PRODUCTION7971
FIG. 6. Effect of glutamic acid substitutions for phosphoserines at aa 2428, 2430, and 2433 on virus production and the interaction of NS5A
with the core protein. (A) Alanine or glutamic acid substitutions for serine residues at aa 2428, 2430, and 2433. The numbers indicate amino acid
positions within the polyprotein of the JFH-1 isolate. The names shown on the left represent full-length HCV or N-terminally HA-tagged NS5A
constructs used in this experiment. Amino acid substitutions are marked in bold and underlined. C represents the C terminus. (B) Effect of alanine
or glutamic acid substitutions on virus production. After transfection of in vitro transcripts of the HCV genomes specified in the inset into Huh-7
cells, the cells and the culture supernatants were harvested at the time points given, and the amounts of core protein were determined by core
protein-specific ELISA. Percent core protein release (vertical axis) indicates the percentage of released core protein in relation to total core protein
(the sum of intra- and extracellular core protein) calculated for each time point. Mean values and standard deviations for at least triplicate
experiments are shown. (C) Effect of alanine or glutamic acid substitutions on the interaction between NS5A and the core protein. N-terminally
FLAG-tagged core protein and N-terminally HA-tagged NS5A carrying defined mutations were coexpressed in Huh-7 cells and immunoprecipi-
tated with anti-FLAG antibody. The resulting precipitates were examined by immunoblotting (IB) using anti-HA or FLAG antibody. One-tenth
of the cell lysates used in IP is as shown as the 10% input.
7972 MASAKI ET AL.J. VIROL.
(50). However, the mechanism by which NS proteins partici-
pate in virus assembly or the role of the interaction between
structural and NS proteins in virus life cycles has not been fully
elucidated. Here, we have clearly demonstrated that HCV
NS5A interacts with the core protein in coimmunoprecipita-
tion experiments not only with coexpression of each epitope-
tagged protein but also with cells expressing the viral genome;
and by using immunofluorescence and subcellular fraction-
ation analysis, we have confirmed that mutations in CL3B
abolish colocalization of NS5A and the core protein, pre-
sumably around LDs. In addition, the intracellular infectiv-
ity assay and IP-RT-PCR strongly suggest that impairment of
the NS5A-core protein interaction results in disruption of virus
production at an early stage of virion assembly. On the basis of
the present results and findings in accompanying articles, one
may infer the following events: newly synthesized HCV RNAs
bound to NS5A are released from the replication complex-
containing membrane compartment and can be captured by
the core protein via interaction with domain III of NS5A at the
surface of LDs or LD-associated membranes. Consequently,
the viral RNAs are encapsidated, and virion assembly proceeds
in the local environment. Recruitment of newly synthesized
viral RNAs to the core protein could be important for efficient
nucleocapsid formation in cells, where concentrations of the
viral genome and the structural proteins are typically low, and
may contribute to the selection of the viral genome to be
packaged. Interaction between NS5A and the core protein has
been previously reported, and the NS5A region containing an
interferon sensitivity determining region and the PKR-binding
sequence (aa 2212 to 2330) has been mapped to that required
for binding with core protein by yeast two-hybrid and in vitro
pull-down assays (13). However, involvement of domain III in
the NS5A-core protein interaction was not analyzed in detail,
and a role for the NS5A-core protein interaction in the HCV
life cycle was not examined in that study.
A growing body of evidence points to phosphorylation of
NS5A as being important in controlling HCV RNA replica-
tion. Although the degree and the requirement for its hyper-
phosphorylation diverge between different HCV isolates, mu-
tations that are associated with increased replicative fitness of
HCV replicons frequently lead to a reduced level of NS5A
hyperphosphorylation (1, 5, 36). Inhibitors of serine/threonine
protein kinases that block NS5A hyperphosphorylation facili-
tate replication of a non-culture-adapted replicon (3, 36). One
model that has been proposed suggests that NS5A hyperphos-
phorylation negatively regulates HCV RNA replication by
disrupting the interaction between NS5A and the vesicle-asso-
ciated membrane protein-associated protein subtype A, a cel-
lular factor considered necessary for efficient RNA replication
(5). However, the regulatory role of the basal phosphorylation
of NS5A in the viral life cycle is poorly understood. It has been
reported that the C-terminal region of NS5A (aa 2350 to 2419)
FIG. 7. Subcellular localization of NS5A and the core protein in HCV-replicating cells. Huh-7 cells were transfected with the in vitro transcript
of the HCV genome, wild type (A) or CL3B/SA (B). Seventy-two hours after transfection, the cells were fixed with 4% paraformaldehyde,
permeabilized with 0.3% Triton X-100, and double stained with antibodies against the core protein (green) and NS5A (red), followed by staining
with an Alexa Fluor 488- or Alexa Fluor 555-conjugated antibody. High-magnification panels are enlarged images of white squares in the merge
panels. (C) HCV (wild type or CL3B/SA)-replicating cells, JFH1/4-1 cells harboring a subgenomic replicon of JFH-1, or Huh/c-p7 cells stably
expressing JFH-1 structural proteins were lysed by freeze-thawing, and the cell lysates were fractionated on 5 to 25% iodixanol gradients. The
distributions of NS5A, calnexin (ER marker), and ADRP (LD marker) were determined by immunoblotting, and those of the core protein were
examined by core protein-specific ELISA.
VOL. 82, 2008 ROLE OF HCV NS5A-CORE INTERACTION IN VIRION PRODUCTION7973
is involved in basal phosphorylation (23). There are highly
conserved serine residues in this region, and alanine substitu-
tions or in-frame deletion of the serine residues has been
shown to impair basal phosphorylation but not to affect RNA
replication in the genotype 1b isolate (1). Consistently, a met-
abolic32P labeling experiment in the present study demon-
strated that NS5A mutants of the JFH-1 isolate in the region
impair the basal phosphorylation. Nevertheless, Tellinghuisen
et al. noted that the serine at aa 2433 of JFH-1 is involved in
generating hyperphosphorylated NS5A, as shown by Western
blotting (50). The basis for this difference is uncertain. To date,
there is no clear evidence to determine which serine residues
located in domain III are phosphoacceptor sites or whether
these residues influence NS5A phosphorylation in an indirect
fashion. Future study to map phosphoacceptor sites in the
NS5A domain III by biochemical approaches is needed.
We found that two of the three serine residues at CL3B are
responsible for regulating the interaction of NS5A with the
core protein as well as for infectious virus production. To
further evaluate the effect of constitutive serine phosphoryla-
tion at the cluster, we replaced the serine residues with glu-
tamic acid, which mimics the presence of phosphoserines. The
S2428/2430E mutant led to restoration of the interaction of
NS5A with the core protein and virus production up to levels
similar to the wild type. Somewhat unexpectedly, the triple
glutamic acid substitution (CL3B/SE) exhibited only a slight
restoration effect or none at all. It is considered that the degree
of negative charge on the glutamic acid residue is not com-
pletely equivalent to that of phosphoserine. It is likely that the
range of acidity at the local environment of the NS5A domain
III that will allow interaction with the core protein is rather
narrow. Induction of a conformational change in NS5A by the
incorporation of phosphate may also be important for its in-
teraction with the core protein. Tellinghuisen et al. reported
that a single serine-to-alanine substitution at aa 2433 blocks
the production of infectious virus and that casein kinase II
likely phosphorylates the residue (50). Although this seems
inconsistent with our results, these investigators also showed
that deletions producing a lack of all three serine residues in
the cluster inhibited virus production more severely than a
single mutation. We observed that a single substitution of
S2428A, S2430A, or S2433A resulted in a moderate decrease
FIG. 8. IP-RT-PCR of HCV-replicating cells performed to examine the association between the core protein and the HCV genome RNA.
Huh-7 cells were transfected with the in vitro transcript of the HCV genome (wild type or CL3B/SA) and lysed in 500 ?l of hypotonic buffer at
72 h posttransfection. After IP with an anti-core protein antibody or mouse IgG, immunoprecipitates were eluted in 100 ?l of elution buffer. RNAs
in immunocomplexes were isolated by acid guanidinium thiocyanate-phenol-chloroform extraction. PCR was carried out as described in Materials
and Methods with primer sets amplifying the fragments of nt 129 to 2367 and nt 7267 to 9463 of the JFH-1 genome. One-tenth (10 ?l) of each
eluted immunoprecipitate was used for assays of the core protein amounts to ensure IP efficiency (lower panel). RNA extracted from a small
aliquot of each cell lysate used in IP-RT-PCR is shown as the input.
7974 MASAKI ET AL. J. VIROL.
in the virus released from the transfected cells; however, more
evident perturbation was obtained from double or triple sub-
stitutions (Fig. 5A and B). Tellinghuisen et al. determined the
HCV production at 48 h after RNA transfection and found a
marked inhibition by the single substitution S2433A. In our
study, as indicated in Fig. 5A, the reduction caused by the
S2433A mutant was approximately 90% at 48 h after transfec-
tion; however, the virus production from the mutant reached a
similar level to that of the wild type at 96 h posttransfection.
Several previous studies have found that apolipoproteins B
(apoB) and E (apoE), microsomal triglyceride transfer protein,
and HCV p7 protein are key factors for production of the
infectious HCV particles (4, 11, 16, 22, 47). Assembly and
maturation of the viral particles appear to depend on the
formation of very-low-density lipoprotein, a large particle con-
taining apoB, apoE, and large amounts of neutral lipids in
hepatic cells. p7 protein is primarily involved in a late step of
virus particle production, and the findings support the idea that
p7 acts as viroporin, which has the capacity to compromise cell
membrane integrity and thus favors the release of viral prog-
eny. How the early step in virion production regulated by the
NS5A-core protein interaction links with the later step(s) in-
volved in the very-low-density lipoprotein assembly or p7 func-
tion remains an interesting question to be addressed.
In summary, we demonstrated that the C-terminal serine
cluster of NS5A (aa 2428, 2430, and 2433), which is involved in
generating the basal phosphorylated form, is a determinant of
NS5A interaction with the core protein and the subcellular
localization of NS5A. Mutation of this cluster blocks the
NS5A-core protein interaction, resulting in perturbation of
association between the core protein and HCV RNA. It is thus
tempting to consider that NS5A plays a key role in transporting
the viral genome RNA synthesized by the replication complex
to the surface of LDs or LD-associated membranes, where the
core protein localizes, leading to facilitation of nucleocapsid
formation. Structural analysis of the NS5A domain III-core
protein complex should provide greater insight into the mode
of interaction between these viral proteins. Identification of
residues at the interface that are involved in important inter-
actions will be of significant value in designing novel structure-
based inhibitors to block the early step of HCV particle for-
We are grateful to Francis V. Chisari (The Scripps Research Insti-
tute) for providing Huh-7 cells. We thank M. Matsuda, S. Yoshizaki, T.
Shimoji, M. Kaga, and M. Sasaki for technical assistance and T.
Mizoguchi for secretarial work.
This work was supported by Grants-in-Aid from the Ministry of
Health, Labor and Welfare; by the Program for Promotion of Funda-
mental Studies in Health Sciences of the Organization for Drug ADR
Relief, R&D Promotion and Product Review of Japan (grant ID:01-3);
by the Japan Society for the Promotion of Science; and by Research on
Health Sciences focusing on Drug Innovation from the Japan Health
Sciences Foundation, Japan. T.M. is the recipient of a Research Res-
ident Fellowship from the Foundation for Promotion of Cancer Re-
search in Japan.
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