JOURNAL OF VIROLOGY, Nov. 2010, p. 10999–11009
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 21
Ultrastructural and Biophysical Characterization of Hepatitis
C Virus Particles Produced in Cell Culture?
Pablo Gastaminza,1*† Kelly A. Dryden,2† Bryan Boyd,1Malcolm R. Wood,3
Mansun Law,1Mark Yeager,2,4,5and Francis V. Chisari1
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 920371; Department of
Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-08862;
TSRI Core Microscopy, The Scripps Research Institute, La Jolla, California 920373; Department of Cell and
Molecular Biology, The Scripps Research Institute, La Jolla, California 920374; and Division of
Cardiovascular Diseases, Scripps Clinic, La Jolla, California 920375
Received 10 March 2010/Accepted 18 July 2010
We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced
in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (?40- to 75-nm
diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their
surfaces. Electron cryomicroscopy revealed two major particle populations of ?60 and ?45 nm in diameter.
The ?60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially
separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed
a high infectivity-to-HCV RNA ratio. The ?45-nm particles lacked a membrane bilayer and displayed a higher
buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-
density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution
ultrastructural information of particle populations displaying differential biophysical properties and specific
infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV
RNA, and viral antigens suggests that infectious particles are likely to be present in the large ?60-nm HCV
particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understand-
ing the structural characteristics of infectious HCV particles.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis
worldwide, with approximately 170 million humans chronically
infected. Persistent HCV infection often leads to fibrosis, cir-
rhosis, and hepatocellular carcinoma (27). There is no vaccine
against HCV, and the most widely used therapy involves the
administration of type I interferon (IFN-?2?) combined with
ribavirin. However, this treatment is often associated with se-
vere adverse effects and is often ineffective (53).
HCV is a member of the Flaviviridae family and is the sole
member of the genus Hepacivirus (43). HCV is an enveloped
virus with a single-strand positive RNA genome that encodes a
unique polyprotein of ?3,000 amino acids (14, 15). A single
open reading frame is flanked by untranslated regions (UTRs),
the 5? UTR and 3? UTR, that contain RNA sequences essen-
tial for RNA translation and replication, respectively (17, 18,
26). Translation of the single open reading frame is driven by
an internal ribosomal entry site (IRES) sequence residing
within the 5? UTR (26). The resulting polyprotein is processed
by cellular and viral proteases into its individual components
(reviewed in reference 55). The E1, E2, and core structural
proteins are required for particle formation (5, 6) but not for
viral RNA replication or translation (7, 40). These processes
are mediated by the nonstructural (NS) proteins NS3,
NS4A, NS4B, NS5A, and NS5B, which constitute the mini-
mal viral components necessary for efficient viral RNA rep-
lication (7, 40).
Expression of the viral polyprotein leads to the formation of
virus-like particles (VLPs) in HeLa (48) and Huh-7 cells (23).
Furthermore, overexpression of core, E1, and E2 is sufficient
for the formation of VLPs in insect cells (3, 4). In the context
of a viral infection, the viral structural proteins (65), p7 (31, 49,
61), and all of the nonstructural proteins (2, 29, 32, 41, 44, 63,
67) are required for the production of infectious particles,
independent of their role in HCV RNA replication. It is not
known whether the nonstructural proteins are incorporated
into infectious virions.
The current model for HCV morphogenesis proposes that
the core protein encapsidates the viral genome in areas where
endoplasmic reticulum (ER) cisternae are in contact with lipid
droplets (47), forming HCV RNA-containing particles that
acquire the viral envelope by budding through the ER mem-
brane (59). We along with others showed recently that infec-
tious particle assembly requires microsomal transfer protein
(MTP) activity and apolipoprotein B (apoB) (19, 28, 50), sug-
gesting that these two components of the very-low-density li-
poprotein (VLDL) biosynthetic machinery are essential for the
formation of infectious HCV particles. This idea is supported
by the reduced production of infectious HCV particles in cells
that express short hairpin RNAs (shRNAs) targeting apoli-
poprotein E (apoE) (12, 30).
HCV RNA displays various density profiles, depending on
the stage of the infection at which the sample is obtained (11,
58). The differences in densities and infectivities have been
* Corresponding author. Present address: Department of Cellular
and Molecular Biology, Centro Nacional de Biotecnologia (CNB-
CSIC), Darwin 3, Madrid 28049, Spain. Phone: 34 915 854 4561. Fax:
34 915 854 506. E-mail: email@example.com.
† P.G. and K.D. contributed equally to this work.
?Published ahead of print on 4 August 2010.
attributed to the presence of host lipoproteins and antibodies
bound to the circulating viral particles (24, 58). In patients,
HCV immune complexes that have been purified by protein A
affinity chromatography contain HCV RNA, core protein, tri-
glycerides, apoB (1), and apoE (51), suggesting that these host
factors are components of circulating HCV particles in vivo.
Recent studies using infectious molecular clones showed
that both host and viral factors can influence the density profile
of infectious HCV particles. For example, the mean particle
density is reduced by passage of cell culture-grown virus
through chimpanzees and chimeric mice whose livers contain
human hepatocytes (39). It has also been shown that a point
mutation in the viral envelope protein E2 (G451R) increases
the mean density and specific infectivity of JFH-1 mutants (70).
HCV particles exist as a mixture of infectious and noninfec-
tious particles in ratios ranging from 1:100 to 1:1,000, both in
vivo (10) and in cell culture (38, 69). Extracellular infectious
HCV particles have a lower average density than their nonin-
fectious counterparts (20, 24, 38). Equilibrium sedimentation
analysis indicates that particles with a buoyant density of ?1.10
to 1.14 g/ml display the highest ratio of infectivity per genome
equivalent (GE) both in cell culture (20, 21, 38) and in vivo (8).
These results indicate that these samples contain relatively
more infectious particles than any other particle population.
Interestingly, mutant viruses bearing the G451R E2 mutation
display an increased infectivity-HCV RNA ratio only in frac-
tions with a density of ?1.1 g/ml (21), reinforcing the notion
that this population is selectively enriched in infectious parti-
The size of infectious HCV particles has been estimated in
vivo by filtration (50 to 80 nm) (9, 22) and by rate-zonal cen-
trifugation (54 nm) (51) and in cell culture by calculation of the
Stokes radius inferred from the sedimentation velocity of in-
fectious JFH-1 particles (65 to 70 nm) (20). Previous ultra-
structural studies using patient-derived material report parti-
cles with heterogeneous diameters ranging from 35 to 100 nm
(33, 37, 42, 57, 64). Cell culture-derived particles appear to
display a diameter within that range (?55 nm) (65, 68).
In this study we exploited the increased growth capacity of a
cell culture-adapted virus bearing the G451R mutation in E2
(70) and the enhanced particle production of the hyperpermis-
sive Huh-7 cell subclone Huh-7.5.1 clone 2 (Huh-7.5.1c2) (54)
to produce quantities of infectious HCV particles that were
sufficient for electron cryomicroscopy (cryoEM) analyses.
These studies revealed two major particle populations with
diameters of ?60 and ?45 nm. The larger-diameter particles
were distinguished by the presence of a membrane bilayer,
characterized by electron density attributed to the lipid head-
groups in its leaflets. Isopycnic ultracentrifugation showed that
the ?60-nm particles are enriched in fractions with a density of
?1.1 g/ml, where optimal infectivity-HCV RNA ratios are ob-
served. These results indicate that the predominant morphol-
ogy of the infectious HCV particle is spherical and pleomor-
phic and surrounded by a membrane envelope.
MATERIALS AND METHODS
Cells and viruses. Hyperpermissive Huh-7.5.1 cell subclone Huh-7.5.1 clone 2
cells (54, 69) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–
10% fetal calf serum (FCS) at 37°C and 5% CO2. Cell culture-adapted JFH-1
variant D183 virus was previously described (70).
Determination of viral infectivity titers and HCV RNA copy number. Infec-
tivity titers were determined in Huh-7 cells by endpoint dilution and immuno-
fluorescence, as previously described (20, 69). Infectivity titers are expressed as
the number of infection focus forming units per ml (FFU/ml).
HCV RNA levels were determined in total cellular RNA extracted by the
guanidinium thiocyanate-phenol-chloroform (GTC) method (13) followed by
quantitative reverse transcription-PCR (RT-qPCR), as previously described
(69). To determine HCV copy numbers, standard curves were prepared by serial
10-fold dilution of a known amount of a plasmid bearing the amplified HCV
Virus production, concentration, and purification. Huh-7.5.1. cells (3.5 ? 106
cells/flask) were plated in T162 vented cap cell culture flasks (Corning, Lowell,
MA). The next day, cells were inoculated at a multiplicity of 0.01 with D183 virus
stocks and incubated at 37°C. Supernatants were collected after 3 days and
replaced with 15 ml/flask of serum-free OptiMem medium (Invitrogen, Carlsbad,
CA). After an additional 20 h of incubation, the supernatants were collected, the
cells were replenished with 15 ml of warm OptiMem and incubated for an
additional 20 h, and then the final supernatants were collected. The supernatants
from days 4 and 5 were filtered through a 0.45-?m-pore-size filter and stored at
Crude virus supernatants, typically ?300 ml (from 20 T162 flasks), were
concentrated in Centricon centrifugal devices with a 100-kDa cutoff (Millipore,
Temecula, CA) by centrifugation at 3,500 rpm until they reached the desired
final volume (?1 ml). Concentrated samples were overlaid onto a 1-ml 20%
sucrose-TNE (10 mM Tris-HCl, pH 8, 150 mM NaCl, 2 mM EDTA) layer
floating over a cushion of 1 ml of 60% sucrose-TNE (Fig. 1A). Samples were
subjected to ultracentrifugation in an SW60 rotor for 2 h at 150,000 ? g. Four
fractions were collected by aspiration from the top, and most of the infectivity
and HCV RNA were concentrated in fraction 3, which corresponded to the 20 to
60% sucrose interphase (Fig. 1A). Sucrose was removed from fraction 3 by
exchange into TNE buffer by centrifugation in Microcon-100 centrifugal devices
with a 100-kDa cutoff (Millipore, Temecula, CA).
Purification of total virus populations by sedimentation velocity. Cushion-
purified virus samples were diluted in 200 ?l of TNE buffer and overlaid onto a
continuous (10 to 50%) sucrose gradient (3.6 ml) (Fig. 1B). Rate-zonal ultra-
FIG. 1. Infectious HCV particles purified by sucrose gradient cen-
trifugation. (A) Concentrated supernatants isolated from infected cells
were subjected to ultracentrifugation on a sucrose step gradient.
(B) The particles collected from the 20–60% interface (fraction 3)
were further purified in a continuous 10 to 50% sucrose gradient.
Infectivity and HCV RNA levels were determined by titration and
quantitative PCR, and the results were expressed as the numbers of
FFU per ml and HCV RNA genome equivalents (GE) per ml,
11000 GASTAMINZA ET AL.J. VIROL.
centrifugation at 200,000 ? g for 1 h separated particles in the supernatant by
virtue of their sedimentation velocity (20). Twelve fractions (?300 ?l) were
collected from the top and analyzed for viral infectivity and HCV RNA. Most of
the infectivity and HCV RNA were typically located in fractions 5 and 6 (Fig.
1B), which were pooled, buffer exchanged into phosphate-buffered saline (PBS),
and concentrated into small volumes (?25 ?l) by ultrafiltration through Micro-
con-100 filters (Millipore, Temecula, CA).
Purification of viral particles by isopycnic ultracentrifugation density. Cush-
ion-purified, concentrated virus samples (?200 ?l) were overlaid onto a discon-
tinuous gradient with 20, 30, 40, 50, and 60% sucrose steps (?700 ?l) and
subjected to overnight ultracentrifugation at 120,000 ? g, as previously described
(20). Twelve fractions were collected from the top and analyzed for buoyant
density, viral infectivity, and HCV RNA content. The highest infectivity-HCV
RNA ratio was detected in fractions whose density ranged from 1.10 to 1.14 g/ml.
These fractions were pooled, buffer exchanged into PBS using Microcon filters as
described above, and compared with pooled fractions of lower (?1.1g/ml) and
higher (?1.4 g/ml) densities, which displayed substantially lower specific infec-
tivity than the intermediate-density fractions.
Virus immunoprecipitation experiments. Agarose beads (?50 ?l) containing
the monoclonal neutralizing recombinant human IgG against HCV E2 (AR3A)
(36) or the anti-HIV recombinant human IgG (B6) (52) were kindly provided by
Mansun Law (The Scripps Research Institute, La Jolla, CA). The beads were
equilibrated in PBS and incubated overnight at 4°C with ?250 ?l of crude virus
preparations in OptiMem (diluted 1:5 in PBS). As a control, diluted virus was
incubated in the absence of beads to determine the baseline infectivity and HCV
RNA values. After overnight incubation, the supernatant was collected and
tested for infectivity. RNA was extracted from washed (three times with PBS)
beads using GTC (4.2 M guanidine thiocyanate, 0.75 M sodium citrate, pH 7.3,
0.5% sarcosyl) (13), and the HCV RNA copy number was determined as de-
Negative-stain electron microscopy. Samples (25 ?l) were fixed by addition of
1 volume of 8% paraformaldehyde (PFA) in 0.1 M cacodylate buffer (pH 7.4).
After incubation for 20 min, aliquots (4 to 5 ?l) were placed on Parafilm (Alcan
Packaging-Neenah, WI). Parlodion-coated nickel grids were subjected to glow
discharge and then immediately inverted onto the droplets. After incubation for
3 min at room temperature (RT), the grids were blotted, and negative staining
was performed by application of 3% uranyl acetate (pH 4) for 2 min at RT. The
stain was removed by blotting, and the grids were air dried. Grids were examined
using a transmission electron microscope (Philips FEI CM100; Eindhoven, Neth-
erlands) operating at 100 kV. Digital images were generated by direct recording
using a charge-coupled-device (CCD) camera (SIS Megaview III camera; Olym-
pus, Munster, Germany) or by scanning micrographs (Finescan 2750 high reso-
lution scanner; Fujifilm, Edison, NJ).
Immunolabeling. Sample grids were inverted onto a PBS–5% bovine serum
albumin (BSA) droplet for 5 min at RT and then incubated for 1 h at RT with
human monoclonal anti-E2 AR3A antibody (36) and/or a mouse monoclonal
anti-apoE antibody (clone 3D12; Meridian Life Science, Inc., Memphis, TN)
diluted 1:20 and 1:10 in PBS–1% BSA, respectively. The grids were then washed
three times (2 min each time) with PBS–1% BSA, followed by incubation for 1 h
at RT with the corresponding secondary antibodies conjugated to 6- or 12-nm
gold particles (Jackson Labs, Watson Grove, PA) at various dilutions in PBS–1%
BSA. The grids were then washed with PBS, fixed for 5 min in PBS–1% glutar-
aldehyde, washed with H2O for 5 min, and stained and imaged as described
Detergent treatment. Purified particles were fixed with 1% glutaraldehyde for
20 min at RT. One aliquot was treated with one volume of 0.4% NP-40 in PBS
for 5 min, and a second aliquot was treated with detergent-free PBS. Both
samples were then purified through a 20% sucrose cushion, buffer exchanged
into PBS, and then negatively stained as described above.
Viral antigen quantification. Relative core protein levels were determined by
enzyme-linked immunosorbent assay (ELISA) as previously described (20). Rel-
ative E2 levels were quantified by ELISA using a recombinant anti-E2 mono-
clonal IgG (AR3A) (36). Briefly, gradient fractions were adsorbed overnight at
4°C onto a Nunc Maxisorp ELISA plate (Thermo Fisher Scientific, Rochester,
NY). Wells were washed twice with 200 ?l of PBS and fixed for 20 min at RT with
4% PFA in PBS (pH 7). Wells were washed twice and blocked with 5% nonfat
milk in PBS for 1 h at RT. Wells were then washed twice with 200 ?l of PBS and
replenished with 50 ?l of a 2 ?g/ml dilution of the recombinant anti-E2 IgG
(AR3A) (36) in binding buffer (3% BSA–0.3% Triton X-100 in PBS). Wells were
washed four times with 200 ?l of PBS and replenished with 50 ?l of an 80 ng/ml
dilution of horseradish peroxidase (HRP)-conjugated goat anti-human antibod-
ies (Pierce, Rockford, IL). After incubation for 1 h at RT, wells were washed four
times with PBS before the developing reagent was added (Pierce, Rockford, IL).
The colorimetric reaction was stopped by adding 1 volume of 1 M H2SO4. Serial
2-fold dilutions of the peak fraction were used to generate a standard curve that
was used for conversion of the values of the optical density 450 nm (OD450).
Electron cryomicroscopy. For biosafety, highly concentrated virus prepara-
tions were fixed in PFA-cacodylate buffer as described above to inactivate viral
infectivity. Purified HCV samples were vitrified by standard methods for cryoEM
(66). In brief, an aliquot (?3 ?l) was applied to a glow-discharged, perforated
carbon-coated grid (either 2/4 Cu-Rh Quantifoil or 2/2-4C C-flat), blotted with
filter paper, and rapidly plunged into liquid ethane. Low-dose images were
recorded at a magnification of ?50,000 on an FEI Tecnai F20 Twin transmission
electron microscope operating at 120 kV, with a nominal underfocus ranging
from 1.5 to 3.5 ?m and a pixel size of 0.271 nm at the specimen level. All images
were recorded with a Gatan 4,000- by 4,000-pixel CCD camera utilizing the
manual mode of Leginon data collection software (62). The grids were main-
tained at ?180°C using a Gatan 626 cryo-stage.
Image analysis. Image analysis was performed using the NIH open source
software Image J (available at: http://rsbweb.nih.gov/ij/). Particle diameters were
measured on digitized images, both point to point by ruler and by comparison
with circles of defined radii. Analysis was performed only on intact particles, with
fewer than 1% of the particles appearing to be broken.
Classification of particle types into zero, one, or multiple membrane bilayers
was based on morphological analysis of the cryoEM images by two or more
independent observers. Only particles displaying a distinct electron-dense bilayer
were considered enveloped (E). Nonenveloped (NE) particles did not display a
visible bilayer. A minor population of large vesicular (LV) particles was observed
that consisted of enveloped particles whose diameters were larger than 85 nm.
Finally, a minor multivesicular (MV) particle population was observed that
displayed two or more clearly distinguishable bilayers.
For illustration, raw images were resized and processed using the Gaussian
blur algorithm (radius, 2 to 3 pixels) in Adobe Photoshop CS2 (version 9.0.2)
Statistical analysis. Statistical significance of the difference between the mean
diameters of particles with different buoyant densities was calculated using a one
tailed, two-sample t test assuming unequal variance (heteroscedastic).
Preparation of highly purified HCV particles. The low yield
of infectious HCV in cell culture has impeded ultrastructural
and biochemical analysis. This barrier has been partially over-
come by the development of a robust cell culture system. By
the use of a cell culture-adapted (D183) virus with vigorous
growth characteristics (70) and a hyperpermissive cell clone
derived from Huh-7.5.1 cells (54), the yield of infectious virus
was increased by two logs from a titer of ?104to ?106FFU/
ml. Infection of Huh-7.5.1 clone 2 (Huh-7.5.1 c2) cells at a low
multiplicity of infection ([MOI] 0.01) produced stocks that
routinely contained 2 ? 106to 5 ? 106FFU/ml 3 days after
infection. Substitution of complete medium with serum-free
OptiMem (Invitrogen, Carlsbad, CA) 20 h before collection of
the supernatants did not reduce the yield of infectious virus
(data not shown), and high-titer (?106FFU/ml) virus stocks
with reduced serum content could be generated. This enabled
efficient concentration of infectious supernatants by ultrafiltra-
tion through anisotropic membranes (Centricon filters with a
100-kDa cutoff; Millipore, Temecula, CA), resulting in virus
preparations containing ?108FFU/ml and nearly 1011HCV
We purified the concentrated viral particles by ultracentrif-
ugation as described in Materials and Methods. Briefly, con-
centrated supernatants were pelleted through a 20% sucrose
cushion onto a 60% cushion (Fig. 1A). Partially purified par-
ticles were collected from the 20–60% interface, where most
(?75%) of the infectivity and HCV RNA were recovered (Fig.
1A, fraction 3). This material was subjected to ultracentrifu-
gation in a continuous 10 to 50% sucrose gradient to purify
VOL. 84, 2010cryoEM ULTRASTRUCTURE OF HCV PARTICLES11001
viral particles based on their sedimentation velocities (Fig. 1B).
This procedure permitted recovery of virtually all the infectiv-
ity (?85%) and HCV RNA in a single peak (Fig. 1B, fraction
5), which migrated with the expected sedimentation velocity
(20). In contrast to what we observed for the parental JFH-1
virus (20), the majority of both the infectivity and the HCV
RNA of the D183 virus comigrated in the same fraction (Fig.
1B, fraction 5), probably due to the higher average density of
the infectious D183 virus particles reflecting a point mutation
(G451R) in the viral E2 protein (70).
By negative-stain EM, virus-like particles (VLPs) were ob-
served only in the samples from infected cells (Fig. 2) and not
in parallel, control samples from noninfected cells (data not
shown). These preparations revealed a pleomorphic particle
population with various diameters (58.7 ? 19.3 nm; n ? 450)
(Fig. 2), similar to those previously described for JFH-1 virus
produced in cell culture (23, 65) and from infected patients
(42, 57, 64). The particles displayed a smooth surface with no
visible surface projections (Fig. 2).
In order to confirm that these preparations contained HCV
particles, we incubated them with a well-characterized E2-
specific neutralizing antibody (AR3A) (36) known to specifi-
cally immuno-deplete viral infectivity and immunoprecipitate
HCV RNA from infectious supernatants (see Fig. S1 posted at
A small but significant fraction of the particles (5 to 20%)
displayed specific decoration with 12-nm gold-conjugated sec-
ondary antibody only after incubation with anti-E2 antibodies
(Fig. 2C). It is noteworthy that the antibody-labeled particles
were morphologically indistinguishable from those not deco-
rated (see Fig. S2 posted at http://www.scripps.edu/wieland
/data/Gastaminza/FigureS2.tif). No decoration was observed
with isotype control antibody (data not shown) directed against
the HIV envelope (52), confirming the specificity of the inter-
action and the viral nature of the purified particles. Since we
failed to specifically decorate the particles with different anti-
bodies against the core protein, the major structural compo-
nent of HCV particles, we set out to determine if the purified
particles could be specifically decorated with antibodies against
apolipoprotein E, a cellular factor shown to be associated with
HCV particles in vivo (51) and in vitro (12). Virus-like particles
could be specifically decorated with anti-apoE (12-nm gold)
(Fig. 2D) and anti-E2 (6 nm-gold) (Fig. 2D, white arrows) and
not with an isotype control antibody, reinforcing the notion
that the purified particles display antigens previously found in
When the virus preparation was treated with detergent
(0.2% NP-40), all of the infectivity and most (?80%) of the
HCV RNA content were lost (Fig. 3A), suggesting that the
detergent removed the envelope from the virions and destabi-
lized most of the capsids (46) (Fig. 3A). Negative-stain EM
showed that the detergent-treated particles were smaller in
diameter (45 ? 5 nm; n ? 83) than untreated particles (56.1 ?
10 nm; n ? 145) (Fig. 3B), which recapitulated the decrease in
Stokes radii calculated by sedimentation velocity for patient-
derived particles before and after detergent treatment (51).
Detergent-treated particles also displayed a rough, angular
surface (Fig. 3B, inset) that contrasts with the relatively
smooth surface observed in untreated particles (Fig. 3B, inset).
Negatively stained particles were pleomorphic with an aver-
age diameter of ?55 nm. However, the use of heavy metal
stain (uranyl acetate), a solid support (carbon), and subse-
quent drying would likely result in deformation and loss of
native structure. Thus, we examined the virus preparations by
electron cryomicroscopy, where particles are vitrified in solu-
tion and observed in a close-to-native state.
cryoEM of purified particles. Frozen hydrated particles (Fig.
4) were pleomorphic and similar in appearance to the nega-
tively stained particles (Fig. 2), with an average diameter of
54.6 ? 12.6 nm (range, 31 to ?100 nm; n ? 2,087). The
predominant morphology was spherical, with a relatively
smooth surface devoid of visible projections (Fig. 4). cryoEM
revealed that many of the particles displayed a 5- to 6-nm-thick
electron-dense bilayer (Fig. 4, arrowheads), compatible with a
lipid membrane (25). This feature was used to differentiate the
particle populations into four distinct classes (Fig. 5). Particles
that displayed a visible bilayer were designated enveloped (E),
and particles devoid of this feature were designated nonenvel-
oped (NE) (Fig. 5A). A small number of large vesicles dis-
played one bilayer (LV), and multivesicular particles (MV)
displayed multiple bilayers.
The enveloped and nonenveloped populations were most
abundant in these preparations (94%) and had similar distri-
butions (E, 46%; NE, 53%) while the LV and MV particles
represented only 4% and 1.7%, respectively. Importantly, the
E particles displayed smooth surfaces, and some displayed
clearly discernible internal structures that were separated from
the lipid bilayers. This capsid-like object (Fig. 5) was similar in
size to the NE particles, which were compact and electron
dense, devoid of any internal features or surface projections
(Fig. 5A). LV particles were electron lucent and occasionally
displayed surface projections (Fig. 5A) while MV particles
displayed variable sizes and morphologies and contained het-
erogeneous structures, including internal vesicles and amor-
phous material (Fig. 5A).
These four types of particles displayed different mean diam-
eters (Fig. 5B). The E particles (n ? 880) had a mean diameter
of 60.3 ? 10.4 nm. In contrast, the NE particles had a smaller
diameter with a narrower size distribution (44.24 ? 4.74 nm;
n ? 1,089). Finally, LV and MV particles were much larger
(?85 nm), with extremely variable diameters of 105.6 ? 22.4
nm (n ? 82) and 114.9 ? 31.8 nm (n ? 36), respectively. Four
different virus preparations displayed similar frequencies and
size distributions of the four different particle populations
(see Fig. S3 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS3.tif), which attests to the consistency of
the purification method and analysis.
cryoEM, infectivity, and viral RNA analysis of particles of
different buoyant densities. The previous results show that the
predominant E and NE particle classes isolated from infection
supernatants were associated with most of the HCV RNA and
infectivity. To confirm the viral nature of these particles, we
modified the purification scheme to separate the different par-
ticles based on their buoyant densities. Cushion-purified par-
ticles were separated by ultracentrifugation in isopycnic su-
crose density gradients. Infectivity, HCV RNA, envelope (E2
protein), core protein content, and density were determined
for each fraction as described in Materials and Methods and
Gastaminza et al. (20). The G451R mutation is known to
produce a distinct increase in the average density of infectious
11002 GASTAMINZA ET AL.J. VIROL.
D183 virus particles relative to the parental JFH-1 virus (70).
As expected for the D183 virus, infectivity and HCV RNA
were most abundant in fractions whose density was ?1.15 g/ml
(Fig. 6A, fractions 8 and 9). Also as expected from previous
experiments (16, 20, 21), fractions around 1.1 g/ml (Fig. 6A,
fractions 6 and 7) that preceded the peak of HCV RNA and
infectivity consistently displayed the highest infectivity-HCV
RNA ratio, suggesting that they were relatively enriched in
FIG. 2. HCV particles are pleomorphic and display E2 and apolipoprotein E on their surface. (A) Electron micrograph of highly purified,
negatively stained HCV particles demonstrates their fairly uniform size but pleomorphic shape. Particles were purified as described in the legend
of Fig. 1. Scale bar, 500 nm. (B) Close-up views show the heterogeneous sizes and shapes. (C) Representative images show specific decoration of
a subset (5 to 20%) of particles with an antibody directed against the viral envelope protein E2. (D) Representative images show specific decoration
of E2-positive particles (6-nm gold) with anti-apoE antibodies (12-nm gold). Scale bar, 100 nm (B, C, and D).
VOL. 84, 2010 cryoEM ULTRASTRUCTURE OF HCV PARTICLES11003
infectious versus noninfectious particles (Fig. 6B; see also Fig.
S4 posted at http://www.scripps.edu/wieland/data/Gastaminza
/FigureS4.tif). Therefore, the intermediate fractions preceding
the peak of infectivity likely contain the largest proportion of
infectious particles, albeit in lower numbers.
cryoEM was then used to evaluate particle morphology in
three density pools. The low-density pool (density of ?1.1g/ml)
(low specific infectivity) typically contained less than 2% of the
infectivity and HCV RNA of the preparation. The intermedi-
ate-density pool (?1.10 to 1.14 g/ml) displayed the highest
specific infectivity and contained ?15% of the infectivity and
5% of the total HCV RNA. Finally, the high-density pool
(density of ?1.14g/ml) (low specific infectivity) contained most
of the infectivity (?80%) and HCV RNA (?94%) and most of
the virus-like particles.
As shown in Fig. 6C, the low-density pool that had a low
specific infectivity contained a mixture of E, LV, and MV
particles, with no particular class dominating the population.
In contrast, the intermediate-density pool that had a high spe-
cific infectivity was greatly enriched in E particles, which con-
stituted 49% of the total particle population. The NE, LV, and
MV particles were relatively underrepresented (20, 24, and
6%, respectively). Finally, NE particles dominated the high-
density pool that had a low specific infectivity, representing
85% of the particles in that population, while E particles were
much less frequent (15%). The correlation between the rela-
tive abundance of enveloped particles and the enrichment of
highly infectious particles with the highest infectivity-HCV
RNA ratio was verified in each of the gradients used for the
analysis shown in Fig. 6C (see Fig. S5 posted at http://www
ing the notion that enveloped particles are enriched in samples
with higher specific infectivity. Furthermore, since the frac-
tions enriched in enveloped particles contained high envelope
(E2)/core ratios (see Fig. S6 posted at http://www.scripps.edu
/wieland/data/Gastaminza/FigureS6.tif), it is likely that their
characteristic bilayer corresponds to the viral envelope.
Since infectious HCV particles display heterogeneous buoy-
ant densities (Fig. 6A) and diameters (Fig. 5), we examined
whether the diameters of the enveloped particles were corre-
lated with their buoyant densities. We measured the diameters
of enveloped particles with different densities and observed
small, although statistically significant, differences in the diam-
eters of high-density (57.6 ? 10.1 nm, n ? 225) and interme-
diate-density (63.6 ? 4.5 nm, n ? 260) particles (P ? 2.10?13)
(see Fig. S7A posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS7.tif). These differences in diameters re-
flected a differential size distribution in which there was an
inverse relationship between particle diameter and buoyant
density (see Fig. S7B posted at http://www.scripps.edu/wieland
/data/Gastaminza/FigureS7.tif). Indeed, internal capsid-like
structures could be visualized in enveloped particles of signif-
icantly different diameters in our preparations (see Fig. S7C
The development of a cell culture system for HCV and its
optimization permitted production of high-titer virus stocks.
Purification of secreted particles by sequential ultracentrifuga-
tion yielded virus preparations containing most of the infec-
tious and noninfectious HCV RNA-containing particles.
Negative-stain EM and cryoEM analysis revealed a hetero-
geneous particle population with a mean diameter of ?55 nm
(Fig. 2), similar to patient-derived virus analyzed by rate-zonal
ultracentrifugation (51) and negative-stain EM (33, 37, 42, 57,
64). Detergent treatment resulted in total loss of infectivity and
a measurable reduction in the average virus particle diameter
from ?56 to ?45 nm (Fig. 3), again similar to detergent-
treated, patient-derived particles (51).
cryoEM analysis revealed an electron-dense bilayer around
some particles (Fig. 4). This feature allowed us to distinguish
two morphologically distinct particle classes (enveloped and
nonenveloped) that had different diameters (?60 versus ?45
nm, respectively), were approximately equal in frequency, and
accounted for over 90% of the total particle population (Fig.
5). Previous EM studies of patient sera described particles with
similar diameters (64), indicating that the cell culture-derived
virus recapitulates the morphologies of wild-type virus from
The ?60-nm E-type particles display a 5- to 6-nm thick
surface bilayer (Fig. 4) that most likely represents a lipid mem-
brane (25) and is consistent with removal of the envelope and
reduction in particle diameter with detergent treatment (Fig.
3B). The E particles were present in all of the infectious sam-
FIG. 3. Particles treated with 0.2% NP-40 are noninfectious and
display a decrease in diameter. Detergent-treated and untreated par-
ticles were subjected to ultracentrifugation in a continuous 10 to 50%
sucrose gradient. (A) Detergent treatment eliminated infectivity and
resulted in a substantial loss of HCV RNA. (B) Particle diameter
decreased from ?60 to ?45 nm with detergent treatment. Inset shows
representative negatively stained particles. Scale bar, 50 nm.
11004 GASTAMINZA ET AL.J. VIROL.
ples we analyzed, and they were the dominant population in
preparations that displayed the highest specific infectivity (i.e.,
infectivity-RNA ratios) and E2-core protein ratio (Fig. 6; see
also Fig. S6 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS6.tif). Collectively, our results suggest
that infectious HCV particles are likely present within the E
particle population in these preparations. This is reinforced by
the fact that the average diameter of these D183 particles is
similar to the diameter of infectious JFH-1 virus particles (65
to 70 nm), which we previously calculated from their sedimen-
tation coefficient (20). In addition, it is compatible with the
diameter of infectious HCV estimated by size-based filtration
and inoculation into chimpanzees (8, 22).
The second major particle population (nonenveloped parti-
cles) is devoid of an observable bilayer (Fig. 5) and displays the
average diameter of detergent-treated particles (?45 nm), as
determined by EM (Fig. 3) and rate-zonal ultracentrifugation
(51). Unfortunately, detergent treatment reduced the number
of particles observable by cryoEM so that it was not possible to
compare their morphologies to those of untreated NE parti-
cles. In contrast to the E particles, which are relatively more
abundant in the highly infectious intermediate-density frac-
tions (Fig. 6), the NE particles are relatively more abundant in
high-density fractions that contain most of the viral RNA but
display relatively low infectivity (Fig. 6). Collectively, these
results suggest that the NE particles contain HCV RNA but
are unlikely to be infectious.
The multivesicular (MV) particles and large vesicles are
infection related since they are not found in control samples,
but they barely contribute to total HCV infectivity. Interest-
ingly, they display a striking resemblance to human exosomes
observed by cryoEM (56). Consistent with this hypothesis,
HCV structural proteins are associated with circulating exo-
somes in HCV-infected patients (45). Although MV particles
display very low infectivity in cell culture (Fig. 6), they could
conceivably play a role in natural infections. Additional exper-
iments aiming to determine the nature, biochemical composi-
tion, and functional properties of these exosome-like struc-
tures will be required to evaluate their functional relevance
during natural HCV infection.
The results presented in this study suggest that the bilayer
that differentiates E from NE particles constitutes the viral
FIG. 4. cryoEM showed that purified HCV particles are predominantly spherical and consist of subpopulations with different diameters. Note
that the large-diameter particles are surrounded by an envelope (arrowheads), which is not detectable in the small-diameter particles. Scale bar,
VOL. 84, 2010 cryoEM ULTRASTRUCTURE OF HCV PARTICLES11005
envelope. First, the E particles are found predominantly in
preparations containing the highest E2-core protein ratio
(see Fig. S6 posted at http://www.scripps.edu/wieland/data
/Gastaminza/FigureS6.tif). Second, treatment of purified HCV
particles with detergent, which causes loss of viral envelope
and infectivity, results in a diameter shift from 60 to 45 nm
(Fig. 3), coinciding with the diameters of the E and NE parti-
cles, respectively (Fig. 5). If this is the case, infectious HCV
particles appear to contain an ?45-nm nonicosahedral capsid
that is not tightly associated with the viral envelope. These
features are clearly different from those of other members of
the family. In other members of the Flaviviridae family, the
viral envelope is usually not as distinct by cryoEM, where the
lipid bilayer is tightly associated with the capsid and appears
buried under a thick, smooth glycoprotein layer (34, 35). The
heterogeneous enveloped population with various diameters
FIG. 5. Purified virus preparations contained two major (enveloped and nonenveloped) and two minor (large vesicle and multivesicular)
subpopulations. (A) Gallery of representative electron cryomicrographs showing the four particle classes. Scale bar, 100 nm. (B) Histogram and
table of 2,086 particles from four independent virus preparations revealed roughly similar numbers of enveloped and nonenveloped particles, with
minor populations of large vesicles having a uniform interior density, and a few multivesicular particles that are probably nonviral and may
11006 GASTAMINZA ET AL.J. VIROL.
surrounding an internal capsid resembles porcine reproductive
and respiratory virus ([PRRSV] Arteriviridae) particles (60).
Given the similarity in the sizes of the internal capsid and the
population of nonenveloped particles, it is possible that the NE
particles are capsids lacking an envelope. In fact, NE particles
accumulate in high-density fractions where core antigen levels
are the highest and the E2-core ratio is the lowest (see Fig.
S6 posted at http://www.scripps.edu/wieland/data/Gastaminza
It is unlikely, but formally possible, that the NE particles
display a viral envelope tightly associated with the capsid and
not visible in individual images. Indeed, a recent three-dimen-
sional reconstruction of noninfectious HCV virus-like particles
produced in insect cells suggested that ?50-nm particles have
an envelope tightly associated with the capsid (68). In that
study, these particles were compared with JFH-1 particles pu-
rified from isopycnic gradient fractions that contain the peak of
HCV RNA, have a diameter of 50 nm, and are devoid of any
evident bilayer (similar to particles in our high-density frac-
tions) (Fig. 6). Unfortunately, the morphological heterogene-
ity, relatively low particle yield, and lack of measurable sym-
metry of both E and NE particles precluded the image analysis
and reconstruction necessary to determine if the 45-nm NE
particles display a lipid bilayer.
We are grateful to Takaji Wakita (National Institute of Infectious
Diseases, Tokyo, Japan) for kindly providing the infectious JFH-1
molecular clone, Dennis Burton (The Scripps Research Institute, La
Jolla, CA) for providing the recombinant human anti-E2 and anti-HIV
IgGs, and Michael Houghton (Chiron) for providing the MS3 anti-
serum against HCV core. We thank Marlene Dreux, Urtzi Garaigorta,
Ken Takahashi, Stefan Wieland, and Barbie Ganser-Pornillos for their
expert advice and useful discussions. We are grateful to Erick Giang,
Christina Whitten, and Josan Chung for excellent technical assistance.
This work was supported by NIH grants R01-CA108304 (F.V.C.),
R01-AI079043 (F.V.C.), R01-A79031 (M.L.), and R01-GM066087
This is manuscript number 20607 from the Scripps Research Insti-
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