JOURNAL OF VIROLOGY, Mar. 2011, p. 2138–2147
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 5
Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes
for Single-Cycle Infection by Providing Homologous Envelope
Proteins or Vesicular Stomatitis Virus Glycoproteins in trans?†
Rui Li,1Yan Qin,1Ying He,1Wanyin Tao,1Nan Zhang,1Cheguo Tsai,2Paul Zhou,2and Jin Zhong1*
Unit of Viral Hepatitis1and Unit of Antiviral Immunity and Genetic Therapy,2Key Laboratory of Molecular Virology and
Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences,
Chinese Academy of Science, Shanghai 200025, China
Received 5 November 2010/Accepted 6 December 2010
Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the
major components of viral particles. Here we developed a trans-complementation system that allows the
production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted
(HCV?E). The lack of envelope proteins could be efficiently complemented by the expression of homologous
envelope proteins in trans. HCV?E production could be enhanced significantly by previously described adap-
tive mutations in NS3 and NS5A. Moreover, HCV?E could be propagated and passaged in packaging cells
stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interest-
ingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of
HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production.
VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the
delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the
production of single-cycle infectious HCV particles, which should be useful for studying individual steps of
the HCV life cycle and may also provide a new strategy for HCV vaccine development.
Hepatitis C virus (HCV) is a major etiological agent of
severe liver diseases, including liver cirrhosis and hepatocellu-
lar carcinoma, with an estimated 170 million people infected
worldwide (2). No vaccine is available to prevent HCV infec-
tion, and the sole therapeutic treatment available, based on
interferon, does not always lead to cure and is often associated
with significant side effects (12). HCV is an enveloped plus-
strand RNA virus belonging to the family Flaviviridae. The
9.6-kb viral genome encodes a single polyprotein that is co- or
posttranslationally cleaved into structural (core, E1, and E2)
and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and
NS5B) proteins (4). The structural proteins encapsidate the
viral genome into infectious particles and mediate the entry of
the virus into permissive cells; the nonstructural proteins NS3,
NS4A, NS4B, NS5A, and NS5B are the viral components of
the membrane-bound replication complexes that catalyze
genomic RNA replication (18).
HCV envelope glycoproteins E1 and E2 are processed by
cellular signal peptidases in the endoplasmic reticulum (ER),
are highly glycosylated in the amino-terminal ectodomains,
and are anchored to the membrane by the carboxyl-terminal
transmembrane domains to form a stable noncovalent het-
erodimer complex. This oligomer is thought to be the prebud-
ding form of the functional HCV glycoproteins and is essential
for interaction with the receptor during HCV entry (52). Ac-
cording to the current model for HCV assembly and secretion,
HCV core particles containing the genome assemble in the
lipid droplets and acquire the viral envelope by budding into
the ER (43), during which time the E1 and E2 proteins are
inserted into the viral envelope (15, 45).
The development of HCV infection models that reproduce
the entire HCV life cycle in vitro has created an opportunity to
study each viral protein as a determinant of virus production
(31, 53, 57). A number of recent studies have demonstrated
that besides the structural proteins core, E1, and E2, the non-
structural proteins NS3 and NS5A, as well as p7 and NS2, also
play important roles in virus assembly and secretion (3, 25, 32,
36, 37, 41, 48). Moreover, accumulating evidence suggests that
the association between HCV and host low-density lipoprotein
(LDL) or very low density lipoprotein (VLDL) is important for
virus egress and that apolipoprotein E (apoE), a component of
LDL/VLDL, is required for HCV infectivity and production
(5, 9, 10, 23, 49). These results indicate that HCV assembly and
release are mediated by a concerted interplay between viral
structural proteins, nonstructural proteins, and host factors.
trans-complementation systems have been utilized as a re-
verse-genetics approach for studying the roles of individual
HCV proteins in the viral life cycle independently of their
cis-acting effects. For instance, HCV core protein with lethal
mutations could be rescued by ectopic expression of wild-type
or C-terminally truncated core proteins (28, 36). HCV ge-
nomes with a deletion in p7 could be rescued by the expression
of p7 either with or without the leading signal sequence (8).
Mutations in NS2 blocking virus assembly could be rescued by
expression of NS2 in trans from a helper replicon (24, 56).
Mutations in NS5A domain III disrupting virus production
* Corresponding author. Mailing address: Institut Pasteur of Shang-
hai, Chinese Academy of Science, 225 South Chongqing Road, Shang-
hai, 200025, China. Phone: (86) 21-63858685. Fax: (86) 21-63859365.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 15 December 2010.
could be rescued by a helper replicon expressing functional
NS5A (3). HCV subgenomic replicon RNA lacking the entire
region encoding the structural proteins could produce infec-
tious viruses upon expression of the structural proteins in trans
from helper viruses, stably expressing cell lines, or transient
plasmid transfection (1, 22, 33, 40, 47). However, complemen-
tation of HCV with a deletion of the envelope gene by enve-
lope glycoproteins provided alone in trans has not been re-
Here we developed a trans-complementation system based
on HCV envelope glycoproteins that allows the production
of single-round infectious HCV particles (HCV?E). The lack
of the envelope could be complemented by the expression of
envelope proteins in trans from transient plasmid transfection
or in packaging cells stably expressing HCV envelope proteins
alone. HCV?E could be propagated and passaged in the pack-
aging cells while resulting in only single-round infection in
wild-type cells. In addition, we observed that vesicular stoma-
titis virus glycoproteins (VSV-G) could rescue the production
of HCV lacking endogenous envelope proteins. Further char-
acterization of these pseudotype viruses (HCVvsv) revealed
that HCVvsv entry was indeed mediated by VSV glycoproteins
and that HCVvsv secretion did not require apoE. The wide
host range of VSV glycoprotein-mediated infection would al-
low for the bypass of the natural HCV entry process and the
delivery of HCV replicon RNA into HCV receptor-deficient
cells. Taken together, our development provided a new tool for
producing single-cycle infectious HCV particles, which should
be useful in the study of particular steps of the HCV life cycle.
This technology may also provide a new strategy for the estab-
lishment of HCV replicon cell lines and vaccine development.
MATERIALS AND METHODS
Cells and viruses. The hepatic cell lines (Huh7 and Huh7.5.1) were main-
tained in complete Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal calf serum, 10 mM HEPES buffer, 100 U/ml penicillin, and 100
mg/ml streptomycin. Huh7.5.1E packaging cells were produced by transfecting 2
?g of the pcDNA3-JFH1-E1/E2 plasmid into 8 ? 105Huh7.5.1 cells, followed by
3 weeks of selection with 500 ?g/ml G418. The cell clone with the highest E2
protein expression levels was expanded and used for the studies. To generate an
apoE knockdown cell line, a lentiviral vector encoding short hairpin RNA
(shRNA) targeting apoE (5?-AGACAGAGCCGGAGCCCGA-3?) was cotrans-
fected with plasmids encoding compatible packaging proteins and VSV-G into
HEK293 cells, as described previously (14). At 72 h posttransfection, cell super-
natants were collected, filtrated, and used to transduce Huh7.5.1 cells. A control
cell line expressing shRNA targeting firefly luciferase (5?-CGTACGCGGAAT
ACTTCGA-3?) was generated in the same way.
Plasmids. Plasmids pUC-JFH1-delE and pFGR-JFH1-delE, which contain an
in-frame deletion in the regions of JFH1 encoding E1 and E2, have been de-
scribed previously (11, 53). Plasmid pcDNA3-JFH1-E1/E2, expressing JFH1
envelope glycoproteins, was constructed by PCR amplification of the JFH1 E1
and E2 regions (amino acid residues 171 to 750) and insertion of the PCR
product into pcDNA3.1. The plasmids encoding the envelope proteins of other
HCV strains were generated similarly. Plasmid pLP/VSV-G, expressing the gly-
coproteins of vesicular stomatitis virus, was obtained from Invitrogen (Carlsbad,
CA). All plasmids constructed were verified by DNA sequencing.
Indirect immunofluorescence. Intracellular immunostaining was performed as
described previously (57). Briefly, the cells were fixed with 4% paraformaldehyde
and were permeabilized with 0.5% Triton X-100. HCV E2, core, NS5A, and
VSV-G were stained by using a human monoclonal anti-E2 antibody (C1) (17),
a mouse monoclonal anti-core antibody (C7-50; Abcam, Cambridge, United
Kingdom), a rabbit polyclonal anti-NS5A antibody (a generous gift from Kuni-
tada Shimotohno, Kyoto University, Kyoto, Japan), and a monoclonal anti-
VSV-G antibody (P5D4; Abcam), respectively. Bound primary antibodies were
detected by using Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary
antibodies (Molecular Probes, Eugene, OR). Nuclei were stained with
Western blot analysis. Cells were collected in radioimmunoprecipitation assay
(RIPA) buffer (150 mM NaCl, 50 mM Tris [pH 8], 1% NP-40, 0.5% deoxy-
cholate, and 1% sodium dodecyl sulfate [SDS]) and were quantified by a bicin-
choninic acid (BCA) assay (Pierce, Rockford, IL). Cell lysate proteins were
separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and were
then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford,
MA). Membranes were probed first with a primary antibody against HCV E1
(made in-house), E2 (Biodesign International, Saco, ME), NS3 (8G-2; Abcam),
or ?-actin (Sigma, St. Louis, MO) and then with alkaline phosphatase-conju-
gated goat anti-rabbit, donkey anti-goat, or goat anti-mouse secondary antibod-
ies (Promega, Madison, WI). Proteins were visualized by a 5-bromo-4-chloro-3-
indolyl-?-D-galactopyranoside (BCIP)–nitroblue tetrazolium kit (Promega).
HCV infectivity titer and RNA quantification. HCV infectivity titers were
determined with Huh7.5.1 cells by endpoint dilution and immunostaining as
described previously (57). HCV RNA levels were determined by quantitative
reverse transcription-PCR (RT-PCR) as described previously (17).
Density gradient ultracentrifugation. Gradients were formed by overlaying 2
ml of 20%, 30%, 40%, 50%, and 60% sucrose solutions in TNE buffer (10 mM
Tris-HCl [pH 8], 150 mM NaCl, 2 mM EDTA) as described previously (17).
Equilibrium was reached by ultracentrifugation for 16 h at 30,000 rpm
(154,000 ? g) in an SW41 Ti rotor at 4°C in a Beckman Optima L-80XP
preparative ultracentrifuge. Fifteen gradient fractions of 750 ?l were collected
from the top, and their infectivity titers and HCV RNA levels were determined
as described above. The density of each fraction was determined by measuring
the mass of 100-?l aliquots of each sample.
HCV infection kinetics assay. Eighty thousand Huh7.5.1 or Huh7.5.1E cells
were seeded into 12-well plates, left overnight, and then inoculated with the virus
at a multiplicity of infection (MOI) of 0.01. The infected cells reached confluence
on day 4 postinfection and were then split at a ratio of 1:3 into 12-well plates
(harvested on day 6), 6-well plates (harvested on day 8), and T25 flasks (har-
vested on day 10). Culture supernatants were collected at the time points indi-
cated in the figures, and infectivity titers were determined as described above.
Transwell-based trans-complementation assay. Eighty thousand Huh7.5.1E
cells were seeded in 12-well plates, left overnight, and then inoculated with
HCV?E at an MOI of 0.05. On day 4 postinfection, the infected cells were
collected and reseeded at 2 ? 104/cm2on a permeable membrane (pore size, 1.0
?m) in the upper chamber of a transwell system (BD Falcon; Bedford, MA), and
naïve Huh7.5.1 cells were seeded at 2 ? 104/cm2in the lower chamber of the
transwell system. After the percentage of infected Huh7.5.1 cells reached more
than 50% at day 6 postcoculture, the infected Huh7.5.1 cells were collected and
reseeded at 2 ? 105/well in 24-well plates for the transfection of HCV or VSV
glycoproteins using Lipofectamine 2000 (Invitrogen) according to the manufac-
turer’s protocol. The culture supernatants were collected at day 3 posttransfec-
tion, and viral infectivity was determined.
Preparation of anti-VSV-G serum. We used virus-like particles (VLP) express-
ing VSV-G as an immunogen to generate anti-VSV-G serum. To produce VLP,
4.5 ? 106HEK293T cells were cotransfected with 14 ?g of pCMV?R8.2 (38) and
10 ?g of the VSV-G plasmid using a calcium phosphate precipitation method.
The VLP-containing supernatants were harvested at 16 h posttransfection,
loaded onto a 20% sucrose cushion, and ultracentrifuged at 20,000 rpm for 2.5 h
at 4°C in a Beckman SW28 rotor. The pellets were resuspended in phosphate-
buffered saline (PBS) and were stored at ?80°C until use. To generate anti-
VSV-G immune sera, female BALB/c mice were injected intraperitoneally with
total 200 ?l VLP expressing VSV-G in both the prime and boost injections
(separated by a 3-week interval). Seven days after the boost, serum samples were
collected, heat inactivated at 56°C, and stored in aliquots at ?80°C.
Blockade of HCV infection. The blockade of HCV infection by a human
monoclonal anti-E2 antibody (C1) (17) or a mouse anti-VSV-G serum was
performed as described previously (49). The infection efficiency was determined
3 days postinfection by counting the number of NS5A-positive foci (cell culture-
grown HCV [HCVcc]) or cells (HCV?E and HCVvsv).
Preparation of MLVpp. Human immunodeficiency virus (HIV)-based murine
leukemia virus pseudoparticles (MLVpp) were generated as described previously
(54). Briefly, 293T cells were cotransfected with plasmids encoding HIV pack-
aging proteins, an HIV vector containing luciferase, and MLV glycoproteins.
Supernatants were harvested at 72 h posttransfection and were filtered. Infection
was quantified by measuring luciferase activity on a GloMax 96 microplate
HCV secretion assay. About 1 ? 105apoE knockdown cells plated in 24-well
plates were infected with the HCV?E virus at an MOI of 0.5. The cells were
washed with warm medium to remove the initial virus inocula on the following
VOL. 85, 2011trans-COMPLEMENTATION OF HCV ENVELOPE BY VSV-G2139
day. Then the cells were transfected with the HCV or VSV envelope glycopro-
teins using Lipofectamine 2000 (Invitrogen). The culture supernatants were
collected at day 3 posttransfection, and viral infectivity and HCV RNA levels
VSV glycoprotein-mediated HCV replicon colony formation assay. HCV rep-
licon cells stably expressing the Neo-labeled JFH1 genome lacking the regions
encoding E1 and E2 were produced by transfecting in vitro transcripts of pFGR-
JFH1-delE into Huh7 cells as described previously (11). The VSV-G expression
plasmids or the empty vector was transfected into the established replicon cells
using Lipofectamine 2000 (Invitrogen). At day 3 posttransfection, culture super-
natants were used to inoculate 1 ? 105naïve Huh7 or CD81-deficient Huh7
(designated R3 ) cells seeded in a 12-well plate. One day postinfection, the
culture medium was replaced with complete DMEM containing G418 (800
?g/ml), which was refreshed every 3 days thereafter. Three weeks after trans-
fection, G418-resistant colonies were stained with crystal violet as described
Virus production by JFH1 with the envelope gene deleted
could be rescued by the expression of envelope proteins in
trans. Previous studies have shown that a defect in HCV pro-
teins such as core, p7, NS2, and NS5A could be complemented
by expressing the missing proteins in trans (3, 8, 24, 25, 28, 36,
56). To test whether the function of HCV envelope glycopro-
teins E1 and E2 could be complemented in trans, we con-
structed JFH1-delE, in which the regions encoding E1 and E2
(from amino acid position 217 to 567) were deleted, and a
helper plasmid expressing the full-length JFH1 E1 and E2
proteins under the control of the cytomegalovirus (CMV) pro-
moter (Fig. 1A). These two cassettes included all HCV non-
structural proteins and cis elements required for HCV RNA
replication and all structural proteins required for viral ge-
nome packaging. Therefore, if these two cassettes are coex-
pressed within the same cell, viral particles containing a defec-
tive HCV genome lacking the envelope genes should be
produced and should be infectious. We cotransfected the
JFH1-delE RNA transcripts with the helper plasmid into
Huh7.5.1 cells. As shown in Fig. S1A in the supplemental
material, both core and E2 proteins could be detected simul-
taneously in a small percentage of transfected cells on day 3
posttransfection. Furthermore, the culture supernatants col-
lected from the cotransfected cells contained infectious viruses
(?80 infectious units/ml; designated HCV?E), while the cul-
ture supernatants collected from cells cotransfected with
JFH1-delE RNA and the empty vector or a plasmid expressing
JFH1 E1 or E2 alone possessed no infectivity (see Fig. S1B in
the supplemental material). Importantly, Huh7.5.1 cells in-
fected with HCV?E expressed HCV core proteins but not E2
proteins (data not shown), indicating that the HCV RNA ge-
nomes packaged in HCV?E indeed lacked the envelope-en-
coding regions. Furthermore, RT-PCR analysis using a primer
set outside the envelope-encoding region confirmed that the
envelope-encoding region was indeed deleted in the HCV?E
genome, as expected (Fig. 1B).
The efficiency of HCV?E production from the cotransfec-
tion of JFH1-delE RNA with the envelope-expressing helper
plasmid was very low (?80 infectious units/ml), likely due to
the low percentage of expression of JFH1-delE RNA and
envelope proteins within the same cell. To improve the effi-
ciency of HCV?E production, we transfected the envelope-
expressing plasmid into Huh7.5.1cells and selected a G418-
resistant cell line that stably expressed JFH1 E1 and E2
proteins. We designated this cell line Huh7.5.1E. Both West-
FIG. 1. JFH1 with a deletion in the envelope genes could be rescued by expression of envelope proteins in trans. (A) Schematic representations
of the JFH1-delE RNA genome, carrying an in-frame deletion of 350 amino acids within the regions encoding E1 and E2, and a helper plasmid
expressing the full-length E1 and E2 proteins. The amino acid positions of deletion sites within the envelope region are indicated as 217 to 567.
(B) RT-PCR analysis of the HCV genome in the supernatants of Huh7.5.1 cells transfected with JFH1-delE RNA and an empty vector (lane 1),
JFH1-delE RNA and the helper plasmid (lane 2), or wild-type JFH1 RNA (lane 3) using a primer set flanking the envelope-encoding region. The
DNA size marker is shown on the right. (C) Detection of E1 and E2 expression in Huh7.5.1E packaging cells by Western blot analysis. Proteins
were separated from Huh7.5.1E cell lysates by 12% SDS-PAGE and were probed with antibodies specific to HCV E1 and E2. The expression of
?-actin was examined as a protein loading control. (D) Kinetics of viral infectivity in the supernatants of naïve Huh7.5.1 and Huh7.5.1E cells after
transfection of JFH1-delE RNA. Broken lines indicate the detection limit of the titration assay. Means and standard deviations from three
independent experiments are shown.
2140 LI ET AL.J. VIROL.
ern blot and immunofluorescence analyses confirmed the ex-
pression of envelope proteins in Huh7.5.1E cells (Fig. 1C; see
also Fig. S2 in the supplemental material). Next, we trans-
fected JFH1-delE RNA into Huh7.5.1E cells. The frequencies
of coexpression of JFH1-delE RNA and envelope proteins
were about 80% (see Fig. S2 in the supplemental material).
Importantly, about 300 infectious units/ml of HCV?E was
detected at 72 h posttransfection, while no virus was produced
when JFH1-delE RNA was transfected into the parental
Huh7.5.1 cells (Fig. 1D).
Taken together, our data showed that the deletion of the
envelope genes of JFH-1 could be rescued by the expression of
the envelope E1 and E2 proteins in trans from transient plas-
mid transfection or in packaging cells stably expressing these
Adaptive mutations enhanced HCV?E production. It has
been shown that cell culture-adaptive mutations, especially
those in nonstructural proteins, could enhance HCVcc produc-
tion (27, 55). To further improve the efficiency of HCV?E
production, we engineered two previously described cell cul-
ture-adaptive mutations, M1051T in NS3 and C2219R in
NS5A, into JFH1-delE. Both mutations were selected from a
cell culture with a persistent long-term infection (58) and have
been shown to enhance HCVcc production (Y. He and J.
Zhong, unpublished data). The wild-type and double mutant
JFH1-delE RNAs were electroporated into Huh7.5.1E cells.
As shown in Fig. 2A and B, the HCV RNA and NS3 protein
levels from day 1 to day 3 posttransfection were similar for
wild-type and double mutant JFH1-delE, indicating compara-
ble transfection and HCV genome replication efficiencies.
However, as shown in Fig. 2C, the introduction of two muta-
tions into the JFH1-delE genome dramatically improved
HCV?E production. The infectious viruses could be detected
as early as 24 h posttransfection and reached a peak titer of 5
? 103infectious units/ml at 48 h posttransfection. Thus,
HCV?E produced from JFH1-delE with these two mutations
was used in subsequent studies.
HCV?E could propagate in the packaging cells but resulted
in only single-cycle infection in naïve cells. Next, we tested
whether HCV?E produced from the RNA transfection exper-
iment could propagate in Huh7.5.1E packaging cells. For this
purpose, we infected Huh7.5.1E and Huh7.5.1 cells at an MOI
of 0.01 with HCV?E viral supernatants collected from the
transfection experiment for which results are shown in Fig. 2,
and we monitored virus production at the time points postin-
fection indicated in Fig. 3. As shown in Fig. 3A, HCV?E
resulted in productive infection in Huh7.5.1E cells, with am-
plification kinetics very similar to those of HCVcc with the
same adaptive mutations in Huh7.5.1E cells, but produced no
infectious viruses in Huh7.5.1 cells. Furthermore, we showed
that HCV?E could be passaged in Huh7.5.1E cells multiple
times without losing infectivity (data not shown).
To further confirm the single-cycle-infection nature of
HCV?E, we tested the abilities of HCV?E and HCVcc to
form foci of infection in Huh7.5.1 or Huh7.5.1E cells, since the
formation of foci of infected cells is an important marker for
productive HCV infection, as previously reported (57).
HCV?E and HCVcc were serially diluted and inoculated into
either Huh7.5.1 or Huh7.5.1E cells. The cells were fixed 3 days
later and were assayed for HCV core protein expression by
immunofluorescence. As shown in Fig. 3B, HCVcc infection
resulted in the formation of foci of infected cells (?20 core-
positive cells per focus) in both Huh7.5.1 and Huh7.5.1E cells,
as expected, whereas HCV?E infection resulted in focus for-
mation only in Huh7.5.1E cells, not in Huh7.5.1 cells (?6
infected cells per infection origin, likely due to cell division
during 3 days of infection), indicating that HCV?E was unable
to produce infectious progeny viruses in Huh7.5.1 cells to in-
fect adjacent cells.
Taken together, these results demonstrated that HCV?E
could propagate and be passaged in the packaging cells but
produced only single-cycle infection if no HCV envelope pro-
tein was provided in trans.
Rescue of HCV?E production with heterologous HCV enve-
lope proteins or vesicular stomatitis virus glycoproteins. Next,
we tested whether HCV?E production could be rescued by
envelope proteins of different HCV strains or a different virus.
For this purpose, we used a transwell-based infection system to
deliver the JFH1-delE genome into naïve Huh7.5.1 cells. As
shown in Fig. 4A, naïve Huh7.5.1 cells seeded in the lower
chamber of a transwell system (34) were cocultured with
HCV?E-infected Huh7.5.1E cells grown in the upper chamber
FIG. 2. Adaptive mutations enhanced HCV?E production. Huh7.5.1E cells were transfected either with wild-type (wt) JFH1-delE or with the
JFH1-delE mutant (mut) containing M1051T in NS3 and C2219R in NS5A. (A) Analysis of the replication kinetics of the indicated HCV genomes
by quantitative RT-PCR. Means and standard deviations for three independent experiments are shown. (B) Cell lysate proteins were analyzed by
Western blot analysis using anti-NS3 and anti-actin antibodies on days 1, 2, and 3 posttransfection (p.t.). (C) The supernatants were collected at
days 1, 2, 3, and 4 posttransfection, and their viral infectivity was determined by the titration assay. Broken lines mark the detection limit of the
titration assay. Means and standard deviations for three independent experiments are shown.
VOL. 85, 2011 trans-COMPLEMENTATION OF HCV ENVELOPE BY VSV-G2141
for 6 days until more than 50% of naïve Huh7.5.1 cells had
been infected with HCV?E, as demonstrated by HCV core
protein immunostaining (Fig. 4B). Then we withdrew the up-
per chamber and transfected the HCV?E-infected Huh7.5.1
cells in the lower chamber with plasmids expressing the enve-
lope proteins of different HCV strains or foreign glycoproteins
from VSV. Immunofluorescence analysis of HCV E2 and
VSV-G expression on day 3 posttransfection indicated compa-
rable transfection efficiencies (see Fig. S3 in the supplemental
material). Culture supernatants were collected on day 3 post-
transfection and were analyzed for infectivity by the titration
assay. As shown in Fig. 4C, JFH1 and J6 (both genotype 2a
strains) envelope proteins could rescue HCV?E production
(8,000 and 4,000 infectious units/ml, respectively), whereas
H77 (genotype 1a) and Con1 (genotype 1b) envelope proteins
could not. To our surprise, VSV-G was able to rescue HCV?E
production, reaching an infectivity titer of 1,000 infectious
units/ml (these pseudotype viruses are designated HCVvsv).
These data suggested that chimeric HCV?E trans-packaging
could be achieved by providing intragenotypic envelope pro-
teins or foreign envelope proteins (VSV-G) in trans.
HCVvsv entry did not depend on interaction between E2 and
CD81. Next, we determined whether the entry of HCV?E and
HCVvsv was dependent on the molecular interactions between
E2 and CD81. First, we preincubated HCVcc, HCV?E, and
HCVvsv with a human monoclonal anti-E2 antibody (30) or a
mouse anti-VSV-G serum for 1 h prior to infection. As shown
in Fig. 5A, the anti-E2 antibody inhibited infection by HCVcc
and HCV?E but had no effect on infection by HCVvsv. In
contrast, the anti-VSV-G serum efficiently inhibited infection
by HCVvsv but had no effect on infection by HCVcc or
HCV?E (Fig. 5B), clearly demonstrating that the entry of
HCV?E and HCVvsv was mediated by HCV and VSV glyco-
Second, we examined the dependency of HCV?E and
HCVvsv entry on CD81, a critical receptor for HCV. Equal
amounts of HCVcc, HCV?E, and HCVvsv were used to inoc-
ulate Huh7 cells and R3 cells, Huh7 derivatives that lack CD81
expression (58). As shown in Fig. 5C, Huh7 cells were equally
susceptible to infection by the three viruses. However, R3 cells
could be infected only by HCVvsv, not by HCVcc or HCV?E.
These data indicated that HCVvsv infection was no longer
restricted by the normal HCV infection tropism. It has been
FIG. 3. Single-cycle infection of HCV?E in naïve Huh7.5.1 cells.
(A) Infectious kinetics of HCV?E and HCVcc in Huh7.5.1 and
Huh7.5.1E cells. The cells were inoculated with HCVcc or HCV?E at
an MOI of 0.01. Culture supernatants were harvested at the indicated
time points postinfection, and their infectivity was determined by the
titration assay. Broken lines mark the detection limit of the titration
assay. Means and standard deviations for two independent experi-
ments are shown. (B) Immunofluorescence analysis of core proteins
(green) in Huh7.5.1 and Huh7.5.1E cells infected with 100 infectious units
of HCVcc or HCV?E. Nuclei (blue) were stained with Hoechst dye.
FIG. 4. Production of HCV?E containing envelope proteins of dif-
ferent HCV strains or VSV. (A) Schematic drawing of the transwell
system used for coculturing the HCV?E-infected packaging cell line
(upper chamber) with the naïve Huh7.5.1 cell line (lower chamber). Six
days after coculture, the upper chamber was removed, and the cells in
the lower chamber were transfected with plasmids expressing envelope
glycoproteins. (B) The percentage of HCV?E-infected Huh7.5.1 cells
in the lower chamber was determined by core immunofluorescence
analysis at day 6 after coculture. (C) Rescue of HCV?E by transfection
of helper plasmids expressing glycoproteins of different HCV strains or
VSV. The culture supernatants were collected at 72 h posttransfection,
and the viral infectivity of the supernatants was determined by the
titration assay. Means and standard deviations for three independent
experiments are shown.
2142 LI ET AL.J. VIROL.
reported previously that both HCV and VSV enter host cells
through low-pH-dependent endocytosis (7, 16, 20, 35, 42, 50).
To determine whether HCVvsv entry was still dependent on
endocytosis, we tested NH4Cl and bafilomycin A1, inhibitors
that prevent the acidification of endosomal compartments.
HCVcc was used as a control for endocytosis-dependent entry,
while HIV-based pseudoparticles bearing amphotropic murine
leukemia virus (MLVpp) (54), which fuses directly at the
plasma membrane at a neutral pH, was used as a control for
endocytosis-independent entry. As shown in Fig. 5D, infection
by HCVcc, HCV?E, or HCVvsv was effectively blocked by
pretreatment of cells with NH4Cl or bafilomycin A1, while
HIV-MLV infection was not affected. These data suggested
that HCVvsv also requires a low-pH step for productive entry.
Buoyant densities of HCV?E and HCVvsv. Next, we ana-
lyzed the buoyant densities of HCVcc, HCV?E, and HCVvsv
by sucrose density gradient analysis. After ultracentrifugation
in a 20 to 60% sucrose gradient, the infectivity titers and HCV
RNA contents of each density fraction were determined. As
shown in Fig. 6, the infectivities of HCV?E and HCVcc were
distributed over a broad range of density fractions (90% of
infectivity was recovered from 7 fractions between 1.02 and
1.14 g/ml), with a mean density of 1.08 g/ml. Notably, HCV?E
possessed more infectivity and genomic-RNA-containing viral
particles in the low-density fractions (1.01 to 1.05 g/ml) than
HCVcc, perhaps due to the reduced amounts of glycoproteins
in the HCV?E envelope. In contrast, the infectivity of HCVvsv
was distributed over a smaller range of density fractions (90%
of infectivity was recovered from 5 fractions between 1.08 and
1.15 g/ml), with a mean density of 1.10 g/ml. These results
suggested that HCVvsv particles had a more homogeneous and
FIG. 5. Characterization of the entry processes of HCVcc,
HCV?E, and HCVvsv. (A and B) Blockade of infection with different
HCV particles by an anti-E2 (A) or anti-VSV-G (B) serum. Fifty
infectious units of each virus indicated was incubated with a human
monoclonal anti-E2 antibody or a mouse anti-VSV-G serum for 1 h
prior to inoculation. The infection was analyzed by NS5A immunofluo-
rescence analysis 3 days later, and the number of positive foci
(HCVcc) or positive cells (HCV?E and HCVvsv) was expressed as a
percentage of that for the mock treatment control. Error bars repre-
sent the standard deviations from three independent experiments.
(C) The same amounts of HCV particles were serially diluted and were
then inoculated into Huh7 and R3 (CD81?) cells. The infection was
analyzed by NS5A immunofluorescence analysis 3 days later. Broken
lines mark the detection limit. Means and standard deviations for three
independent experiments are shown. (D) Huh7.5.1 cells were either
mock treated (filled bars) or incubated for 1 h with a medium con-
taining 10 mM NH4Cl or 20 nM bafilomycin A1 (Baf-A1) (shaded or
open bars, respectively). Then the cells were washed with the medium
and were infected with different viruses for 4 h in the presence or
absence of the drug. As a control for pH-independent virus entry,
infections with lentiviral pseudoparticles bearing MLV envelope pro-
teins were performed in the same way. The infection was analyzed 3
days later by NS5A immunofluorescence analysis except for MLVpp,
for which infection efficiency was determined by measuring luciferase
activity. Means and standard deviations for three independent exper-
iments are shown.
FIG. 6. Characterization of the buoyant densities of HCVcc,
HCV?E, and HCVvsv. The different HCV particles were subjected to
a 20% to 60% sucrose gradient. Fifteen fractions were collected from
the top, and the infectivity titer and HCV RNA level of each fraction
were determined by the titration assay and quantitative RT-PCR. The
results are expressed as percentages of the totals for all viruses. The
density of each fraction was determined by measuring the mass of a
100-?l aliquot of the fraction. The data shown are representative of
results from two independent experiments.
VOL. 85, 2011trans-COMPLEMENTATION OF HCV ENVELOPE BY VSV-G2143
heavier buoyant density profile than HCV?E and HCVcc,
possibly due to less association of HCVvsv particles with host
apoE was not required for the secretion of infectious HCV-
vsv. It has been demonstrated recently that HCVcc secretion is
associated with the host lipoprotein secretory pathway and that
apoE, an important component of LDL/VLDL, is required for
HCV production and infection (5, 9, 10, 19, 21, 23, 39). To
assess the role of apoE in HCV?E and HCVvsv production,
we established a Huh7.5.1 apoE knockdown cell line, in which
apoE expression was stably downregulated with an apoE-spe-
cific shRNA, and a control cell line that expressed an unrelated
shRNA. The apoE shRNA led to a profound and stable re-
duction in apoE expression (Fig. 7A). Then we verified the
effect of apoE knockdown on HCVcc production. The apoE
knockdown and control cells were infected with HCVcc at an
MOI of 5 as described in Materials and Methods. At 24 h
postinfection, the cells were assayed for intracellular HCV
RNA levels, and the culture supernatants were assayed for
infectivity titers. As shown in Fig. 7B, the HCV RNA levels in
the control and apoE knockdown cells were comparable, while
extracellular infectivity titers were significantly reduced in
apoE knockdown cells. This result was consistent with previous
findings (5, 19, 23), clearly demonstrating that apoE is required
for HCVcc secretion.
Next, we investigated whether HCV?E and HCVvsv secre-
tion also required apoE. As shown in Fig. 7C, apoE knock-
down and control cells were inoculated with HCV?E at an
MOI of 0.5. At 24 h postinoculation, one portion of infected
cells was collected for the determination of intracellular HCV
RNA levels, in order to compare the HCV?E infection effi-
ciencies in the two cell lines, and another portion of cells was
washed extensively with medium to remove the initial inocula
and was then transfected with plasmids expressing the JFH1 or
VSV glycoproteins. Extracellular infectivity and HCV RNA
levels in the supernatants from the transfected cells were de-
termined at 60 h posttransfection. Our results showed that the
intracellular HCV RNA levels for the two cell lines were com-
parable (Fig. 7D). However, as shown in Fig. 7E and F, the
knockdown of apoE expression significantly reduced the infec-
tivity titers and total HCV RNA levels of HCV?E in the
supernatants but did not affect HCVvsv secretion at all. Col-
lectively, these data demonstrated that apoE was required for
HCV?E secretion but not for HCVvsv secretion, suggesting
that HCVvsv egress may not be associated with the host LDL/
VLDL secretory pathway.
Establishment of HCV replicon cells by HCVvsv transduc-
tion. Our data showed that HCVvsv infection was not re-
stricted by the normal HCV infection tropism, raising the pos-
sibility that HCVvsv could be used to deliver HCV RNA
containing an antibiotic-selectable marker into cells that are
nonpermissive for HCV entry. To test this possibility, we first
transfected the envelope gene-deleted JFH1 replicon RNA
containing a neomycin marker (Fig. 8A) into Huh7 cells in
order to establish replicon cells that stably replicated the HCV
genome. The established replicon cells were then transfected
with a plasmid expressing VSV-G. Three days later, the culture
supernatants were transferred to CD81-negative R3 cells in
FIG. 7. Analysis of the dependency of HCVcc, HCV?E, and HCVvsv secretion on apoE. (A) Western blot analysis of apoE expression in apoE
knockdown (shApoE) and control cells. (B) apoE knockdown and control cells were infected with HCVcc at an MOI of 5. Extracellular infectivity
and intracellular HCV RNA levels were determined by the titration assay and quantitative RT-PCR. (C) Protocol for the HCV secretion assay.
apoE knockdown and control cells were infected with HCV?E at an MOI of 0.5. At 24 h postinoculation, one portion of infected cells was collected
for the determination of intracellular HCV RNA levels, and another portion of cells was transfected with a plasmid expressing JFH1 or VSV
glycoproteins (gps). (D) Determination of intracellular RNA levels in order to compare HCV?E infection efficiencies in the two cell lines. (E and
F) The extracellular infectivity (E) and HCV RNA levels (F) of the supernatants from transfected cells were determined at 60 h posttransfection.
The data are presented as percentages of the control level from two independent experiments performed in duplicate. Means ? standard
deviations are shown.
2144 LI ET AL.J. VIROL.
order to select G418-resistant colonies. As shown in Fig. 8B,
the culture supernatants from VSV-G-transfected cells pro-
duced visible colonies after 4 weeks of G418 selection, while
the culture supernatants of cells transfected with the empty
vector did not. The presence of HCV replicon RNA in the
G418-resistant cells was further confirmed by immunostaining
for HCV core (Fig. 8C) and by RT-PCR (data not shown).
In this study, we developed a new way to produce infectious
HCV particles that encapsidate the HCV genome lacking the
envelope-encoding regions (HCV?E). Steinmann and cowork-
ers showed previously that the luciferase-tagged JFH1 genome
lacking the envelope-encoding regions alone could be rescued
by a helper virus expressing the entire JFH1 genome, but virus
production was low as determined by the luciferase assay, and
it was not certain whether this virus lacking the envelope-
encoding regions alone was able to expand and propagate in
packaging cells, although these investigators did show that an
HCV subgenomic replicon could be trans-complemented and
that the viruses produced could be passaged in the packaging
cell line that expressed all HCV structural proteins (47). More-
over, Pacini and coworkers showed that the envelope gene-
deleted J6/JFH1 RNA could be rescued by the full-length
J6/JFH1 genome or by the regions encoding the entire struc-
tural proteins (core-NS2) in trans, but not by the expression of
envelope proteins alone in trans (40). During the revision of
this article, Bianchi and colleagues also showed that the a
E1E2-deficient JFH1 genome could be rescued by a packaging
cell line expressing autologous envelope proteins (6). Our
work was in agreement with their results and provided a more
detailed characterization of these virus particles.
In addition, we found that the envelope proteins of J6CF
(genotype 2a) could rescue JFH1-based HCV?E production
but that the envelope proteins of H77 (genotype 1a) and Con1
(genotype 1b) could not (Fig. 4), possibly due to intergenotypic
incompatibility. Yi and colleagues reported that the adaptive
mutation Q221L in the helicase domain of NS3 could rescue
infectious virus production by compensating for the NS2-me-
diated assembly defect in the H77/JFH1 chimera (32, 55).
However, our results showed that the production of HCV?E
with the Q221L mutation still could not be rescued by H77
envelope proteins (data not shown), suggesting that more mo-
lecular interactions between structural and nonstructural viral
proteins may contribute to this intergenotypic incompatibility.
Steinmann and coworkers also reported that a JFH1 sub-
genomic replicon (NS3-NS5B) could be trans-complemented
by a helper virus expressing Con1 structural proteins, albeit
with low efficiency (47). This result is contrast to our observa-
tion and could be explained by the fact that they provided the
entire structural proteins (core-NS2) in trans from the same
subtype, whereas we provided only the E1 and E2 proteins in
trans. Thus, a more thorough understanding of the network of
viral protein interactions involved in HCV assembly and secre-
tion is needed in order to produce HCV?E containing enve-
lope proteins of a wide range of HCV genotypes.
The most interesting finding of our study is that foreign
envelope glycoproteins (VSV-G) could substitute for HCV
envelope function in viral assembly and entry. To our knowl-
edge, this is the first study to demonstrate that VSV-G could
form an envelope for HCV particles. This was an unexpected
finding, given that HCV envelope proteins have been shown to
be retained mainly on the ER membrane and that nascent
HCV virions bud into the ER lumen, while VSV-G are local-
ized mainly on the plasma membrane (20, 29, 52). Our further
characterization showed that HCVvsv entry was indeed medi-
ated by VSV-G and did not depend on HCV receptors. Inter-
estingly, unlike that of HCVcc and HCV?E, the release of
HCVvsv particles was not affected by the knockdown of apoE,
a critical host factor involved in HCV egress (5, 9, 19, 23). This
may imply that HCVvsv exits cells independently of the host
LDL/VLDL secretory pathway. In agreement with this finding,
HCVvsv particles exhibit a narrower and heavier buoyant den-
sity distribution than HCVcc and HCV?E. It has been shown
that VSV-G could package a Semliki Forest virus (SFV) RNA
replicon to produce virus-like particles, and this process in-
volved the release of intracellular vesicles containing VSV-G
and SFV RNA (13, 44, 46). It is possible that the release of
HCVvsv particles may occur through a similar mechanism.
Further studies will be required to determine the molecular
mechanisms of HCVvsv morphogenesis.
HCVvsv can be used to deliver an HCV genome containing
an antibiotic-selectable marker in order to establish replicon
FIG. 8. Establishment of HCV replicon cells by HCVvsv transduc-
tion. (A) Structure of the JFH1 replicon RNA with the envelope genes
deleted and containing a neomycin-selectable marker. (B) R3 cells
(CD81?Huh7 derivative) were inoculated with culture supernatants of
empty-vector- or VSV-G-transfected neo-JFH1-delE replicon cells.
Inoculated cells were cultured for 4 weeks in a medium supplemented
with G418 (800 ?g/ml), and G418-resistant colonies were stained with
crystal violet. The results shown are representative of two independent
experiments. (C) Immunofluorescence analysis of HCV core (red) in
the established replicon cell clone after HCVvsv transduction. Nuclei
(blue) were stained with Hoechst dye.
VOL. 85, 2011trans-COMPLEMENTATION OF HCV ENVELOPE BY VSV-G 2145
cell lines. As a proof of concept, we showed that VSV-G easily
delivered neo-JFH1-delE RNA into CD81-negative Huh7 cells
and successfully established a neomycin-resistant HCV repli-
con cell line. Our approach has obvious advantages over the
traditional electroporation-based method for establishing
HCV replicon cell lines, since electroporation needs many cells
and in vitro-synthesized HCV replicon RNA transcripts and is
generally toxic to the host cells. Another advantage of the
HCVvsv transduction-based approach is that VSV-G confers a
wide host cell tropism. This is particularly useful in establishing
HCV replicons in nonhepatic cell lines, since it has been shown
that HCV could replicate in HeLa, HEK293, and mouse fibro-
blast cell lines (26, 51).
There is a great need for effective vaccines to prevent HCV
infection. HCV?E producing single-cycle infection in naïve
cells should provide a new means of HCV vaccine develop-
ment. The greatest advantage of HCV?E is that this virus
possesses all the structural viral proteins to induce humoral
immune responses and that, upon infection, it could produce
all the nonstructural viral proteins in host cells to induce cell-
mediated immune responses. Of course, further studies, espe-
cially in animals, are needed to determine the immunogenicity,
safety, and efficacy of an HCV?E-based vaccine against HCV
We thank Francis Chisari (The Scripps Research Institute, San
Diego, CA) for providing Huh7.5.1 and R3 cells, Takaji Wakita (Na-
tional Institute of Infectious Diseases, Tokyo, Japan) for providing the
JFH1 molecular clone, Mansun Law and Dennis Burton (The Scripps
Research Institute) for providing recombinant anti-E2 human IgG,
and Kunitada Shimotohno (Kyoto University, Kyoto, Japan) for pro-
viding the anti-NS5A antibodies.
This study was supported by grants to J.Z. from the MOST 973
program (2009CB522501), the National Key Programs on Infectious
Disease (2008ZX10002-014), the National Natural Science Founda-
tion of China (30870127), the Chinese Academy of Science (KSCX1-
YW-10), the Institute of Pathogen Biology (2008IPB107), the Shang-
hai Pasteur Health Research Foundation (SPHRF2009001), and the
Partenariat Scientifique TOTAL—Institut Pasteur: Recherche & For-
mation pour la Pre ´vention des He ´patites Virales en Chine.
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VOL. 85, 2011trans-COMPLEMENTATION OF HCV ENVELOPE BY VSV-G 2147