Bidirectional Lipid Droplet Velocities Are Controlled by
Differential Binding Strengths of HCV Core DII Protein
Rodney K. Lyn1,2, Graham Hope3, Allison R. Sherratt1, John McLauchlan3*, John Paul Pezacki1,2*
1National Research Council of Canada, Ottawa, Ontario, Canada, 2Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada, 3Medical Research Council-
University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom
Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core
protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how
mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast
imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics.
Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of
movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the
temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD
localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is
accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the
viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.
Citation: Lyn RK, Hope G, Sherratt AR, McLauchlan J, Pezacki JP (2013) Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of
HCV Core DII Protein. PLoS ONE 8(11): e78065. doi:10.1371/journal.pone.0078065
Editor: Ravi Jhaveri, University of North Carolina School of Medicine, United States of America
Received July 12, 2013; Accepted September 9, 2013; Published November 1, 2013
Copyright: ? 2013 Lyn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was funded by the National Research Council of Canada and a grant from the Canadian Institutes for Health Research (CIHR). R.K.L. was
the recipient of an Ontario Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (JPP); firstname.lastname@example.org (JM)
Once thought to be a benign storage organelle, the lipid droplet
(LD) has gained attention for its involvement in many cell
functions including cellular signaling, membrane organization,
and trafficking [1,2,3]. LDs are primarily composed of triglycer-
ides (TG) and cholesterol esters in a hydrophobic core that is
surrounded by a phospholipid monolayer . Additionally, the
LD surface is coated in proteins that facilitate cellular signaling
interactions, control the access of metabolic enzymes, and
influence LD movement within the cell [4,5,6,7].
Biochemical and live-cell imaging analyses have shown LD
movement is microtubule-dependent [8,9,10,11] and is facilitated
by motor proteins that move along microtubules radiating from
the microtubule organizing center (MTOC) . LDs are shuttled
towards the MTOC in a dynein-mediated retrograde (minus-end
motion) manner, while movement away from the MTOC is
mediated by kinesin motors in an anterograde manner (plus-end
motion) [12,13,14,15]. Immunofluorescence studies of peroxi-
somes in Drosophila have demonstrated that both motors are
localized on cargo at the same time , with evidence of
bidirectional LD movement shown in human hepatocytes .
Accordingly, bidirectional LD transport is likely coordinated to
direct net movement to meet cellular demands [11,18].
Productive hepatitis C virus (HCV) infection is tightly linked to
hepatic lipid metabolism and requires direct interactions with LDs
for propagation [19,20]. HCV infection is the leading cause of
liver disease, affecting 2 to 3% of the global population [21,22].
More than half of HCV infections result in dense accumulation of
LDs, a phenotype commonly known as liver steatosis [23,24,25].
There is strong support for a direct relationship between HCV and
LDs, highlighting LDs as a key host organelle involved in
HCV is a single-stranded, positive-sense RNA virus encoding a
polyprotein that is processed into 3 structural and 7 non-structural
proteins (reviewed in ). Of particular interest is the core
protein, which forms the viral capsid, since it also accumulates on
the LD surface [17,34,35]. The mature form of core is generated
through sequential cleavage by two host proteases (Figure 1A)
[36,37,38,39,40]. This mature form of core, which consists of two
domains, termed I and II, translocates from the ER to the LD
surface, with domain II (DII) involved in LD binding [41,42]. The
core-LD interaction is essential in the HCV lifecycle, since its
disruption eliminates viralparticle assembly [39,43,44,45].
Although the fate of lipids contained in the core-bound LDs is
unclear, host lipids are used in virtually every step of the viral
lifecycle and function as viral dependant post-translational
[23,26,30,46,47,48]. Ultimately, these interactions also permit
the establishment of platforms involved in viral assembly through
LD-associated membrane interactions [25,49].
HCV-induced changes in LD morphology and dynamics can be
investigated by coherent anti-Stokes Raman scattering (CARS)
microscopy. CARS is a molecular imaging tool that uses high
excitation laser pulses to enhance the vibrational resonances of
chemical bonds, and can be specifically tuned to generate high-
contrast images of select organelles and/or drug molecules in the
cell [50,51]. As such, the C-H bonds of long fatty acid chains that
hostand viral proteins
PLOS ONE | www.plosone.org1November 2013 | Volume 8 | Issue 11 | e78065
are densely packed in LDs generate excellent signal contrast for
LD imaging [52,53]. Furthermore, since CARS is a label-free
technique, video-rate imaging of LD dynamics is possible without
the use of chemical stains that may perturb the cellular
We have previously shown changes in LD morphology as well
as mobility in human hepatoma cells after 48 hours of expression
of a HCV genotype 3a form of core (core3a) [17,35]. In addition,
core3a expression increased cellular LD volume before the LD
migrated towards the perinuclear region. In this study, we focus on
LD dynamics at an earlier time point of core protein expression
using a GFP-tagged DII of JFH1 core protein (DII-core) to
visualize core protein’s localization on the LD surface , in
addition to LD particle tracking. This method enabled particle
tracking of DII-core bound LDs, along with LDs in non-DII-core
expressing cells, and allowed their rates of transport in the cell to
be monitored. We also evaluated single amino acid mutations in
the LD binding domain of core to determine whether LDs are
dynamically modified by the binding strength of DII-core. Overall,
our findings provide new insight into the effects of HCV core
protein on LD dynamics. Uncovering details of the HCV life cycle
not only expands our understanding of this important pathogen,
but also offers alternative targets for the development of host-
Figure 1. GFP-tagged DII-corewtcolocalizes with LDs. (A) Schematic representation of HCV core protein. Distinct interactions belong to each of
the three core protein domains. The mature and immature forms are also shown, and are generated by the two host proteases: signal peptidase (SP,
blue), and signal peptide peptidase (SPP, red). (B) GFP-tagged DII-corewtcontains the membrane binding domain consisting of two a-amphipathic
helices separated by a hydrophobic loop. (C-D) CARS microscopy imaging of LDs in Huh-7 cells expressing GFP-tagged DII-corewt. All images were
collected approximately 20 hours after Huh-7 cells were transfected with (D) DII-corewtand (C) without DII-corewt, which contained only the
lipofectamine transfection reagent. (C) Lipid volumes measured by voxel analysis for mock Huh-7 cells are shown in the CARS image. (D) CARS
imaging captures DII-corewtinduced LD biogenesis and redistribution towards the perinuclear region. The two values in panel 2 represent the
average LD volume for cells expressing DII-corewt(top value, double asterisks) and non-expressing DII-core cells (single asterisks) within the same
field of view (bottom value) as measured by voxel analysis. The error represents standard error of the mean. The n represents the amount of cells
quantified for LD density. This experiment was conducted under two biological replicates. Panel 4 is a magnified image selected by a region of
interest from the merged image to project a clearer view of colocalization between DII-corewtand LDs. All scale bars represent 10 mm.
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Expression of GFP-tagged DII-corewtIncreases Cellular LD
Changes in LD dynamics that are induced by bound
fluorescently-tagged proteins can be monitored by simultaneous
two-photon fluorescence (TPF) and CARS microscopy. With this
method, the dynamics of both bound and unbound LD
populations can be compared within the same cell and/or field
of view. Previously, we showed that an N-terminal GFP-tagged
construct of DII-core from the JFH1 strain (Figure 1B, DII-corewt)
was capable of LD colocalization . These results are supported
herein by simultaneous TPF and CARS microscopy, which show
DII-corewtretained a colocalized pattern with cytoplasmic LDs in
Huh-7 cells (Figure 1D).
It was also possible to make qualitative and quantitative
comparisons between LD dynamics and mobility in DII-corewt
expressing and non-expressing cells under one single field of view.
We observed a distinct change in LD density for cells expressing
DII-corewt(Figure 1D, single vs. double asterisks), while LDs
observed in non-DII core expressing cells were comparable to
mock Huh-7 cells (Figure 1C & D, single asterisks). Voxel analysis
used to calculate LD density revealed a 3-fold increase in LD
density of DII-corewtexpressing cells to non-expressing cells under
the same field of view (Figure 1D). We also observed a change in
LD localization in cells expressing DII-corewt, with LD clusters
located at the perinuclear region (Figure 1D, arrowheads). Our
images show that clusters of LDs were absent in the mock and
non-DII-core expressing cells (Figure 1C & D, single asterisks).
This suggests that DII-corewtis capable of inducing LD migration
towards the perinuclear region much like full-length core ,
likely by affecting interactions with motor proteins that are
involved in LD motility . Importantly, we observed that GFP
did not disrupt DII-core binding to LDs, indicating that GFP-
tagged DII-core is suitable to study the dynamics of LD mobility.
DII-corewtModulates LD Dynamics when it is bound to
the LD Surface
Since DII-corewtbehaves similarly to naı ¨ve full-length core
protein , we assessed whether the interaction between DII-
corewtand LDs affected LD mobility. DII-corewtexpressing cells
contain populations of naı ¨ve and DII-corewt-bound LDs. By
simultaneous TPF and differential interference contrast (DIC)
imaging, the trajectories of LDs from both populations can be
tracked by following LD movements with and without overlap of
the DII-corewtGFP tag. It is important to note that LD mobility
may potentially be affected by factors including cell passage
number, biological replicate and cell confluency. To circumvent
this, in every experiment that was conducted, the LD measure-
ments acquired from Huh-7 cells expressing DII-corewtwere
directly compared with LD measurements from a mock sample of
the same biological replicate. We have previously shown that LDs
in full-length core expressing cells were motile, but travel at half
the speeds by comparison to mock LDs . With GFP-tagged
DII-corewtexpressing cells, we observed a similar pattern, and
showed that DII-corewtcoated LDs traveled with an average speed
of approximately 40.3 nm/sec compared to LDs in mock-treated
Huh-7 cells, which traveled at 67.2 nm/sec (Table 1). To compare
these values, we divided the average speeds of DII-corewtcoated
LDs by LDs in mock cells and observed a ratio of 0.60. To
illustrate these changes more clearly, a representative image was
captured from a time-course movie (Figure 2A–C, arrowheads)
that tracked spatially unique LDs under different expression
conditions within the same field of view. For example, the general
trajectories of LD mobility for individual DII-corewtcoated and
non-coated LDs in the same cell are illustrated (Figure 2D, box 1
vs box 2, inset 1 vs inset 2). As expected, non-coated LDs travelled
a longer distance. Additionally, LDs in an adjacent non-expressing
cell traveled further than LDs that are bound to DII-corewt
(Figure 2D, box 3 and inset 3). Furthermore, the presence of HCV
non-structural proteins, which are recruited to LDs during the
viral lifecycle and are required to form the membranous web ,
do not affect the ability of DII-corewtto induce changes in LD
speeds and travel distances (Figure S1).
The Binding Strength of DII Dictates the Overall LD Mean
Speeds and Travel Distances
Since DII-corewtcan modulate LD mobility, we postulated that
single amino acid modifications targeting the interaction between
DII-core and LD interface could variably affect LD dynamics. To
evaluate this, we mutated glycine 161 (G161) of the DII second a-
helix to alter hydrophobicity, since it is conserved among all six
HCV genotypes and is predicted to lie within the cytosol-lipid
interface [28,55,56]. We have found that increasing the hydro-
phobicity of residue 161 increases the binding strength, while a
hydrophilic substitution decreased binding strength of DII-core to
LDs (Filipe et al., manuscript in preparation). To ensure that the
DII-core161mutations did not disrupt targeting, we first evaluated
whether these DII-core161mutants colocalized with LDs in Huh-7
cells. As shown in Figure 3, GFP-tagged DII-core161mutants
colocalized with LDs, and induced LD migration to the
perinuclear region, as is observed for DII-corewt. Expression of
these mutants also increased LD volumes 3–5 fold.
To investigate the effect of LD binding strength of DII-core on
LD dynamics, we next determined LD speeds of the G161
mutants. Generally, cells expressing DII-core161mutants with
large hydrophobic side chains had slower LD mean speeds than
wt; however, all were consistently lower than the mean LD speeds
of their mock samples (Table 1). DII-coreG161F, for example,
exhibited approximately half the mean LD speed of mock, with a
ratio calculated to be 0.47. Conversely, mean LD speeds of DII-
coreG161Sand DII-coreG161Awere faster than wt, with both ratios
calculated to be 0.77. The LD mean speeds measured for DII-
core161mutant expressing cells gave a general trend that appeared
to depend upon the binding strength of DII with LDs (Table 1).
Similar to what was observed for DII-corewt, populations of
core-coated and naı ¨ve LDs also exist within DII-core161mutant
expressing Huh-7 cells. Therefore, we can directly measure LD
trajectories of differential travel distances for individual LDs within
the same cell, depending on whether the LD is bound to DII-
core161. We used DII-coreG161Fexpressing Huh-7 cells as a
representative image to evaluate both LD populations (Figure
S2A–C). The trajectories of DII-coreG161Fcoated LDs ultimately
traveled shorter distances compared to non DII-coreG161Fcoated
LDs within the same cell (Figure S2D, box 1 vs box 2, inset 1 vs
inset 2). Correspondingly, LDs in non DII-coreG161Fexpressing
cells also resulted in longer travel distances (Figure S2D, box 3 and
inset 3). These observations show that the LD mean speed and
mean travel distances are affected only upon direct binding to DII-
Lower Frequency of High Velocity Travel Runs and High
Frequency of Pauses Contribute to Slower Mean Speeds
for DII-core Coated LDs
To investigate DII-core’s induced suppression of the mean LD
speed, we explored the frequency of low to high instantaneous
velocities of DII-corewtcoated LDs compared to LDs in mock
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cells. Since dynein and kinesin motors mediate cargo transport in
opposite directions, measured LD velocities can provide informa-
tion about whether the mobility of DII-corewtcoated LDs travel
more frequently towards one direction, and thus, reveal differen-
tial activity between the two motors. The trajectories of individual
LDs were tracked using the center of the nucleus as a fixed point
Figure 2. DII-corewtcoated LDs are particle tracked using simultaneous TPF and DIC microscopy. This is a representative image of DII-
corewtexpressed in Huh-7 cells. Three individual LDs with dissimilar environments were selected (A–C, white arrows), and their trajectories were
measured to calculate the overall distances traveled. (D) A larger DIC image of (B) includes boxes to identify each LD trajectory (inset 1–3). The value
above each box (D) indicates their overall travel distances for (1) DII-corewtcoated LD, (2) non DII-corewtcoated LD within the same cell, (3) and a LD
in an adjacent cell not expressing DII-corewt. Each LD trajectory is magnified to demonstrate the LD track with selective freeze frame time-intervals
representing the LD position at their indicated times. Due to frequent bidirectional movements, the displayed trajectories represent a general
movement path, and does not portray total distance. All of the LDs are tracked according to the same start and end time. All scale bars represent
Lipid Droplet Velocity Changes by HCV Core-DII
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relative to the position of the LD. LD travel runs that were
directed towards the MTOC (retrograde manner) were identified
as negative displacement, while LDs that moved away from the
MTOC (anterograde motion), were identified as having positive
displacement (Figure 4A–B). The differential velocity profiles were
then segregated into low (15.7–50 nm/sec), medium (50–180 nm/
sec), and high velocity (.180 nm/sec) travel runs (Figure 4C–D).
LD particle tracking in both directions revealed that the frequency
of high and medium velocity travel runs for DII-corewtcoated LDs
was lower when compared to LDs from the mock sample
(Figure 4C). This is represented as a ratio for the frequency for
DII-corewtcoated LDs divided by LDs from the mock, with similar
ratios determined for both directions. For example, at high
velocity travel runs, the ratios were calculated to be 0.47 for the
anterograde direction, and 0.48 for the retrograde direction
(Figure 4C). The differential frequencies for the high and medium
velocities were also consistent with DII-corewtcoated LDs in Huh-
7 cells expressing a subgenomic replicon of HCV (Figure 4D).
Therefore, the shorter travel distances of DII-corewtcoated LDs is
reflected in the lower frequency of high velocity travel runs, and is
independent of the presence of non-structural HCV proteins that
are involved in membranous web formation and viral replication.
Next, we investigated the differential velocity profiles for the
DII-core161mutant coated LDs to determine if binding strength is
reflected in the frequency of high velocity travel runs. As shown in
Figure S3, high velocity travel runs were less frequent for DII-
core161mutant coated LDs, with relative differences in ratios
corresponding to their expected LD binding strength. This was
clear for the highest binding strength mutant DII-coreG161F, with
ratios of 0.32 and 0.31 for retrograde and anterograde velocities,
respectively (Figure S3A). Reduced strength of binding to LDs, as
seen with the DII-coreG161Smutant, demonstrated the highest
ratios of 0.95 and 0.83 for retrograde and anterograde velocities,
respectively (Figure S3D). Overall, distances traveled by mutant
DII-core161coated LDs appear to correlate with relative frequency
of high velocity LD travel runs.
While observing LD transport, we found that not only do LDs
travel at various velocities, but they also appear to pause in a
stalled state. We postulated that higher frequencies of LD pauses
can also contribute to smaller mean distances traveled by DII-core
coated LDs. To establish the minimal threshold that would
identify LD movement by active transport, we used a microtubule
depolymerizing drug, nocadazole, which halts motor protein
dependent active transport. We have previously reported that LDs
in cells treated with nocadazole were found to move at
approximately 15.7 nm/sec ; velocities below this threshold
were characterized as pauses (Figure 4, Figure S3), and LD
movement above this speed was placed in a range of low, medium,
or high velocity travel runs. In general, pauses were more frequent
for all DII-core161mutants and DII-corewtcoated LDs compared
to mock controls (Figure S3A–D). Since LDs are often observed to
move back and forth in opposite directions, we further calculated
the frequency of directional switches. We observed no clear trend
that correlated directional switches with LD binding strength (data
not shown). Therefore, the higher pause frequency for DII-core
coated LDs, and not frequency of directional switching, is likely to
contribute to the shortening of mean LD travel distances and
mean LD speed.
DII-core bound LDs Spend Equal amounts of Time
Traveling in both the Retrograde and Anterograde
We have previously used live-cell imaging by CARS and DIC
microscopy to visualize the ability of full-length core protein to
induce LD migration towards the perinuclear region associated
with HCV replication and assembly . Based on these data and
published work by Boulant et al., it was suggested that core may
directly or indirectly favor a molecular motor imbalance by
perturbing the mechanics of one motor over the other . Since
expression of full-length and DII-core induces LD migration
towards the perinuclear region, a molecular motor imbalance
should drive a greater frequency of travel runs in the retrograde
direction. For this reason, we counted the total frequency of travel
runs for one direction that combined low, medium, and high
velocity travel runs. However, the frequency of travel runs for wt
and mutant DII-core coated LDs were similar in both directions
over our four minute time course (Figure S3E). Finally,
directionality of LD travel was assessed against cytoplasmic
location, relative to the nucleus, since DII-core coated LDs were
also observed to be scattered throughout the cell (Figure S4). Cells
were divided into regions, as shown in Figure 5C, with regions
identified as close to the perinuclear region (close), middle of the
cytoplasm (mid), and in the cell periphery (far). However, a trend
was not observed for wild-type and mutant DII-core coated LD
velocities. This suggests that at time of analysis, movement of DII-
core coated LDs travel equally in both directions and is unrelated
to its location in the cell, except when the LDs reach the
perinuclear region. Although our time measurements last approx-
imately four minutes, we have included a large data set and
statistics measured from all regions of the cell. Importantly, we
wanted to measure the movement of LDs at a particular stage
during core expression, before core induces LD accumulation in
the perinuclear region. While it is difficult to normalize our data to
Table 1. Mean speeds and travel distances of DII-core coated LDs compared to LDs in [mock transfected] Huh-7 cellsa.
ConstructDistance traveled (mm)Mean speed (nm/s)Ratio
Wild-type (n=39) [n=138] 9.761 [16.260.6]40.363 [67.262]0.60
G161F (n=51) [n=53]5.860.4 [12.460.7]24.362 [51.763] 0.47
G161L (n=19) [n=60]7.460.8 [12.860.8]30.863 [53.363] 0.58
G161S (n=39) [n=46] 8.960.7 [11.560.6] 36.863 [47.763]0.77
G161A (n=51) [n=50]10.260.9 [13.160.8] 42.264 [54.563] 0.77
aThe mean speeds and overall travel distances of LDs are compared in DII-core expressing Huh-7 cells and LDs in mock cells (enclosed in square brackets). The error
represents standard error of the mean. The n represents the number of LDs from eight or more cells in different fields of view, assessed by particle tracking for both
mutant (enclosed in round brackets) and mock samples (enclosed in square brackets). Live-cell imaging was conducted for duration of four minutes acquiring each
frame at rate of 1.65 sec/frame. To minimize variability for LD speeds, all of the experiments that directly compared DII-core coated LDs to LDs in a mock sample were
observed in cells of the same biological replicate. The ratios were calculated by dividing the mean speed of DII-core coated LDs by LDs in the mock cells.
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48–72 hours during the time span of infection, LD mobility
measurements required video-rate imaging that is attainable over
a shorter time course with averaging of many trials.
LDs at the Extreme Perinuclear Region Demonstrate
Two general localizations of DII-core coated LDs were revealed
by CARS and DIC imaging for wild-type and all the DII-core
mutants: scattered throughout the cell (Figure S5, white arrow-
heads), and tightly aggregated in the perinuclear region (Figure S5,
red arrowheads). Perinuclear LD aggregation was not observed in
Figure 3. Simultaneous CARS and TPF microscopy captures LD changes induced by single amino acid mutations in GFP-tagged DII-
core161expressing Huh-7 cells. All images were collected approximately 20 hours after Huh-7 cells were transfected with (A) DII-coreG161F, (B) DII-
coreG161L, (C) DII-coreG161S, and (D) DII-coreG161A. CARS imaging identifies DII-core161induced LD biogenesis and redistribution towards the
perinuclear region under the expression of all DII-core161mutants (A–D, panel 4, arrowheads). The two values in panel 2 represent the average LD
volume for cells expressing DII-core161(top value, double asterisks) and non-expressing DII-core161cells (single asterisks) within the same field of view
(bottom value) as measured by voxel analysis. The error represents standard error of the mean. The n represents the number of cells that were
quantified for LD density. This experiment was conducted under two biological replicates. Panel 4 is a magnified image selected by a region of
interest from the merged image to project a clearer view of colocalization between DII-core161mutants and LDs. The scale bar represents 10 mm.
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mock cells, where LDs were generally observed to be scattered
throughout the entire cell. Up until this point, particle tracking was
focused on DII-core coated LDs scattered throughout the cell to
investigate dynamics of LD mobility resulting from differential
binding strengths of DII-core. Therefore, using DII-coreG161Aas a
representative image, we measured the velocities of the perinuclear
aggregates (Figure 5G). We found that these large LD aggregates
were tightly localized together and had limited mobility
(Figure 5C&G). In contrast, DII-core coated LDs outside of this
region experienced bidirectional travel runs (Figure 5D–F). These
observations suggest that, although DII-core coated LDs remain
mobile at areas outside the perinuclear region, mobility is
abrogated once they reach the perinuclear region.
Molecular motors, such as dynein and kinesin, function by a
mechanoenzyme core containing ATPase activity that facilitates
active transport along the plus-end (towards cell periphery) and
minus-end (towards nucleus) of microtubules [15,57]. Upon entry,
viruses are capable of binding to molecular motor proteins that
move on microtubules to achieve transport. This is common for
adenovirus, herpes simplex virus (HSV), and human immunode-
ficiency virus (HIV), which have capsid and tegument viral
proteins on the surface of their viral particles that, upon entry into
the cell, bind and travel by motor-induced transport to ensure that
viral particles are properly delivered to specific cellular regions to
establish infection [58,59,60,61,62]. Likewise, HCV is able to
directly use the microtubule network upon cell entry , as well
as indirectly after viral RNA translation whereby viral proteins are
the likely components that mediate interactions with motor
proteins . As such, the binding of HCV core protein to LDs
is critical in manipulating LD transport towards the perinuclear
region, and is required in the early stages of viral assembly .
However, our understanding of core-induced modulation of
dynamic LD trafficking is limited.
DII-core Coated LD Dynamics
LDs migrate towards the perinuclear region as early as 20 hours
post-expression of core protein [17,35]. Based on this evidence, we
aimed to capture the dynamics of LD motions just prior to this
time point at a pertinent stage when LDs are targeted by DII-core
Figure 4. DII-corewtcoated LD velocities measured in naı ¨ve Huh-7 cells and Huh-7 cells stably expressing an HCV subgenomic
replicon. (A–B) Average representative measurement of a much larger data set, LD velocities in retrograde or anterograde directed transport are
measured in Huh-7 cells expressing (A) DII-corewtand (B) mock transfected. The velocity amplitudes at each time point are divided into parameters of,
low, medium, and high velocities for both directions. The pink parameter line is indicated by a paused event, which was determined by obtaining the
average speed of LDs from nocadazole treated Huh-7 cells. (C–D) The frequency of low (15.7 nm/sec –50 nm/sec), medium (50.1 nm/sec –180 nm/
sec), and high velocity (.180.1 nm/sec) measurements, expressed as a percentage, in both directions, are plotted after particle tracking LDs in DII-
corewtexpressing (C) Huh-7 cells, and (D) Huh-7 cells harbouring an HCV subgenomic replicon. The velocities are measured for DII-corewtcoated LDs
in DII-corewtexpressing cells, and LDs from mock cells not expressing DII-corewt. The ratios above each set of columns are calculated by dividing the
frequency for each velocity interval of DII-corewtcoated LDs by their respective mock LDs.
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for viral induced trafficking. In this study, molecular imaging was
used to track LD trajectories in live hepatocytes in the presence
and absence of bound DII-core protein, as well as bound mutant
DII-core having variable binding strength to LDs. As seen with
full-length core , expression of wt and G161 mutant GFP-
tagged DII-core protein colocalized with LDs and induced LD
migration to the perinuclear region (Figure 1D and Figure 3A–D).
Supporting earlier evidence of full-length core’s ability to modulate
LD dynamics , LDs coated with wt and mutant DII-core
showed slower mean speeds and a decrease in mean travel
distances (Table 1). In this work, the ability to directly compare
naı ¨ve and GFP-tagged DII-core coated LDs was imperative in
understanding finer details LD dynamics, such as decreased high
velocity travel runs and more pauses for DII-core coated LDs
compared to LDs in cells not expressing DII-core (Figure 4 and
Mutations in DII-core that impact protein structure and/or LD
affinity influence viral assembly and, hence, virion production
[44,45,64]. Since core mobility, as reflected in its ability to
associate and be released from the LD, appears to be important for
virion assembly , we investigated mobility of LDs bound to
DII-core containing mutations at a glycine residue predicted to lie
at the membrane interface . We have found that mutations at
G161 impacted DII-core LD binding strength with respect to the
hydrophobicity of the residue (Filipe et al., manuscript in
preparation). Similarly, our results revealed a trend in LD mobility
ranging from slower speeds and decreased travel distances when
the LD binding strength of DII-core is increased (Table 1). This
trend of reduced mobility upon increased LD binding appears to
follow the whole residue free energies determined for the transfer
from water to a unilamellar vesicle interface (reported in ).
Furthermore, distances traveled by mutant DII-core161coated
LDs correlated with relative frequency of high velocity LD travel
Figure 5. Tracking LD mobility at distinct locations of the cell. While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-
coreG161Ais a representative image acquired from a large data set. Huh-7 cells expressing DII-coreG161Ais shown as (A) a merged image of DIC and
TPF, and (B) TPF. DII-coreG161Acoated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C)
LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as
close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-coreG161Acoated LD for each of the segregated region, and
the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the
perinuclear region with higher levels of DII-coreG161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org8 November 2013 | Volume 8 | Issue 11 | e78065
runs. These results suggest that the greater the time DII-core
protein spends on the LD surface, the greater LD dynamics
deviate from normal. Consistent with this, Counihan et al. also
observed that core coated LDs decreased in their motility .
Previous mutations in this region of core have been shown to be
critical in viral particle assembly . Our results suggest that
core’s LD binding strength and effect on LD speed may play a role
in virion assembly.
Bidirectional Movement of DII-core Coated LDs
Current models propose that both dynein and kinesin remain
associated with cargo during transport, even if only one motor is
active [12,16,67]. Our data supports bidirectional movement for
both naı ¨ve and DII-core coated LDs, which confirms that both
molecular motors remain bound and functional (Figure 4 and
Figure S3). Bidirectional movement was observed for both wt and
mutant DII-core coated LDs, which suggests that overall
directional movement initiated by core is likely not based on LD
binding strength. Furthermore, the prevalence of retrograde and
anterograde directed transport of DII-core coated LDs was equally
observed (Figure S3E). Since we showed localization of LDs to the
perinuclear region, as well as equal bidirectional movements, it is
unlikely that motor imbalance is the sole cause for core-mediated
LD localization to the perinuclear region of the cell. It is possible
that our imaging experiments over a four minute time course was
too short to adequately capture an imbalance between active
motors. However, since we observed many LDs at different
locations within the cell, it is possible that perinuclear localization
is driven by detachment from microtubules at the destination
rather than a dramatic change in motor protein function.
Perinuclear localization and bidirectional movement is not
limited to core-bound LDs, since it has been shown for other
pathogens as well. For example, Suomalainen et al. demonstrated
that despite observation of bidirectional motions, overall net
movement of newly entered adenovirus particles was directed
towards the perinuclear region . The authors showed that
localization to the perinuclear region was dependent on transient
activation of protein kinase A (PKA) and the p38/mitogen-
activated protein kinase (MAPK) pathway. Similar mechanisms
may be involved in core protein induced perinuclear localization
and bidirectional movement of LDs, since core expression has
been shown to activate the p38/MAPK pathway in hepatocytes
DII-core Limits LD Mobility within the Perinuclear Region
We highlighted important dynamics of core-directed mobility
for DII-core coated LDs, and demonstrated that bidirectional
motion is observed for all other DII-core coated LDs that are
located outside of the critical perinuclear region (Figure 5). This
prompted us to investigate the movement of LDs within
perinuclear regions that also have higher levels of localized DII-
core protein (Figure 5C & G). Our results revealed minimal LD
movement, indicative of exclusively paused or trapped LDs. The
ability of DII-core to limit LD mobility within the perinuclear
region could be the result of molecular motor disengagement,
allowing DII-core coated LDs to accumulate. Indeed, Miyanari
et al. reported that LDs are required at the replication and
assembly sites , where LD accumulation could effectively link
early and late viral assembly stages.
Alternatively, DII-core coated LDs may be stabilized in the
perinuclear region by the recruitment of additional host proteins.
One method of stability could be mediated through hijacking the
autophagic pathway that forms aggresomes within sites of
replication and assembly [72,73]. The accumulation of aggre-
somes sequestered around the MTOC could prevent motors from
binding and result in densely packed LDs that are stabilized
without accessible motor proteins. Alternatively, host proteins that
are recruited to LDs by core protein at the perinuclear regions
may play a role in stabilizing LDs at these sites. Recently, a subunit
of host clathrin adaptor protein complex 2 (AP2M1) has been
shown to bind core by recognizing a highly conserved tyrosine-
based sorting signal along the DII region . These proteins sort
intracellular cargo via a clathrin adaptor and can mediate
endocytic functions. Importantly, interference of the AP2M1-core
interaction prevents viral assembly . The same AP2M1
binding motif was also identified in the region of the viral E1
glycoprotein. Such a feature supports the idea that AP2M1 is
recruited to LDs via DII of core protein, and likely mediates
intracellular trafficking of core to sites of assembly where E1 and
E2 proteins reside, prior to the envelopment of the viral particle.
This engagement can possibly stabilize the LD from being bound
to motor proteins once LDs reach this critical area at the
Aggregation of DII-core coated LDs in the perinuclear region
may also result in disconnection from normal metabolic processes.
The increased LD volume detected in the periphery of wt and
mutant DII-core expressing cells is consistent with a reduction in
core-induced TG turnover in LDs, opposed to TG synthesis, as
previously determined by Harris et al . Based on our
identification of restricted LD mobility of perinuclear aggregates,
it is tempting to hypothesize that this mechanism of aggregation
could further limit normal lipid turnover. We have also recently
shown that DII-core is sufficient to initiate a significant change in
NAD(P)H levels, as measured by fluorescence lifetime imaging,
which may also influence LD biogenesis and localization (71).
In this study, we have revealed insight into HCV core protein’s
dynamic control of LD migration. We showed that DII-core
causes a decrease in LD speed similar to full-length core, with
limited effect on directionality within the time frame of our
experiments. We also found that the binding strength of DII-core
further impacted LD mobility, indicating that DII-core has a
temporal impact on the LD with respect to time associated with
the LD. Moreover, the observed bidirectional transport of DII-
core coated LDs may suggest that additional host proteins are
essential in directing transport of core-coated LDs, and these
potential interactions will be the focus of future studies.
Nevertheless, live-cell imaging has revealed several novel aspects
of core-induced LD mobility.
Materials and Methods
Human hepatoma cells (Huh-7) were grown in DMEM medium
supplemented with 100 nM nonessential amino acids, 50 U/mL
penicillin, 50 mg/mL streptomycin, and 10% FBS (CANSERA,
Rexdale,ON). Huh-7 cells harboring the pFK-I389neo/NS3-39/
5.1 subgenomic replicon were maintained in the same culture
(GIBCO-BRL, Burlington, ON). The pFK-I389neo/NS3-39/5.1
subgenomic replicon was kindly provided by Ralf Bartenschlager
(University of Heidelberg, Germany).
250 mg/mLG418 Geneticin
Overexpression of HCV DII-core Protein
Huh-7 cells were seeded at 8.06104cells/well in borosilicate
Lab-Tek chambers (VWR, Mississauga, ON). After 24 h, at a
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org9 November 2013 | Volume 8 | Issue 11 | e78065
confluency of 60–70%, cells were transfected with plasmids that
expressed wild-type (wt) and mutant DII-core suspended in
transfection media including lipofectamine 2000 (Invitrogen
Canada Inc., Burlington, ON). After 4 h, DMEM in 20% FBS
was added in equal volume to the chambers. Details of the GFP-
construct are described elsewhere .
QuikChange site-directed mutagenesis (Stratagene) and primer
design were performed according to the manufacturer’s guidelines,
and confirmed by sequencing.
Coherent Anti-Stokes Raman Scattering, Two-photon
Fluorescence and Differential Interference Contrast
The CARS microscopy system employed a single femtosecond
Ti:sapphire oscillator as the excitation source, as previously
described [51,52]. An Olympus FV300 laser scanning microscopy
system on an IX71 inverted microscope was utilised for imaging
experiments. A 40x Uapo 1.15NA water immersion objective and
a long working distance 0.55 NA condenser were used. The
FV300 was adapted for TPF. Source was a Coherent Mira 900
Ti:sapphire laser producing pulses of approximately 100 fs at
800 nm wavelength with an 80 MHz repetition rate. Laser
scanning microscopy can be readily adapted to DIC by taking
advantage of the high inherent polarization in most laser sources.
The DIC optics were adjusted as they would typically be for
transmitted light use: with the prisms removed the condenser
polarizer was adjusted to cross with the objective polarizer. For
laser scanning, the analyzer, which is in a fluorescence cube in the
IX71, was removed from the beam path. To optimally align the
polarization of the laser with that of the microscope optics, a 700–
1000 nm achromatic half wave plate (WPA1212 Casix) was placed
in the laser path before entering the FV300 scan-box. The
polarization of the laser was adjusted by rotating this wave plate to
minimize the amount of light collected through the condenser
polarizer. The DIC prisms were inserted and the path and the bias
of the objective prism adjusted to the optimal image.
Particle Tracking of LDs in Huh-7 Cells
Particle tracking of LD motion for both speed and distance was
captured using spot tracker add-on with ImageJ, as previously
described . The spot tracker followed the light shaded halo
contrast of LDs as a result of changes in refractive index captured
by DIC imaging. The measurement of directional motion was
calculated from a fixed reference point in the center of the nucleus
relative to the LD at each frame.
Quantitative Voxel Analysis
Quantitative data from the CARS images was determined using
a voxel counting routine in ImageJ as previously described
[17,76,77]. In each image, multiple cells within the same field of
view were counted to generate an average percentage of lipid
Huh-7 cells stably expressing an HCV subgenomic
replicon. (A) CARS and TPF microscopy captures colocalization
between DII-corewtand LDs, and captures DII-corewt-induced
LD localization at the perinuclear region. Panel 4 is a magnified
image selected by a region of interest from the merged image to
project a clearer view of colocalization between DII-corewtand
LDs. (B) Particle tracking DII-corewtcoated LDs and LDs in mock
cells not expressing DII-corewt. The overall mean travel distance
Particle tracking DII-corewtcoated LDs in
and mean speeds were measured. The ratio is calculated by
dividing the mean speed of DII-corewtcoated LDs by LDs from
the mock sample. The n represents the number of LDs that were
particle tracked. Live-cell imaging was conducted for duration of
four minutes with each frame interval acquired at 1.65 sec/frame.
All scale bars represent 10 mm.
using simultaneous TPF and DIC microscopy. This is a
representative image of DII-coreG161Fexpressed in Huh-7 cells.
Three individual LDs with dissimilar environments were selected
(A–C, white arrows), and their trajectories were measured to
calculate the overall distances traveled. (D) A larger DIC image of
(B) includes boxes to identify each LD trajectory (inset 1–3). The
value above each box (D) indicates their overall travel distances for
(1) DII-coreG161Fcoated LD (2) non DII-coreG161Fcoated LD
within the same cell, (3) and a LD in an adjacent cell not
expressing DII-coreG161F. Each LD trajectory is magnified to
demonstrate the LD track with selective freeze frame time-
intervals representing the LD position at their indicated times. Due
to frequent bidirectional movements, the displayed trajectories
represent a general movement path, and does not portray total
distance. All of the LDs are tracked according to the same start
and end time. All scale bars represent 10 mm.
DII-coreG161Fcoated LDs are particle tracked
expressing DII-core161mutants. (A–D) The frequency of
pauses (,15.7 nm/sec), low (15.7 nm/sec –50 nm/sec), medium
(50.1 nm/sec –180 nm/sec), and high velocity (.180.1 nm/sec)
measurements, expressed as a percentage, in both directions are
plotted for LDs in cells expressing (A) DII-coreG161F, (B) DII-
coreG161L, (C) DII-coreG161A, (D) DII-coreG161S. The ratios above
each set of columns is calculated by dividing the frequency for
each velocity interval of DII-core coated LDs by their respective
mock LDs. (E) The total frequency of retrograde, anterograde, and
pauses were also collected and presented as a fold-change
measurement that compared LDs in all DII-core161mutants with
each of their respective mocks.
LD velocities are measured in Huh-7 cells
regions in Huh-7 cells expressing DII-coremutand in the
mock. The average frequency of low, medium, and high velocity
runs for each direction was calculated for LDs bound to (A) DII-
corewt, (B) DII-coreG161F, (C) DII-coreG161L, (D) DII-coreG161S, (E)
DII-coreG161A. The data was separated according to where the LD
was located at a position that was relative to the nucleus, either at a
close, medium, or far location.
Frequency of LD velocities at three different
was observed. This is a representative image with a pattern that
is typically observed in all other DII-core constructs. The white
arrow represents a LD population of individual LDs that are
bound to DII-coreG161A. The red arrow corresponds to tightly
packed LDs with a high abundance of DII-coreG161Acolocalized
at the same region. Individual LDs are indistinguishable at this
region. All scale bars represent 10 mm.
Two populations of DII-coreG161Acoated LDs
The authors would like to thank A. Ridsdale, D. Moffatt, and A. Stolow for
help with microscopy.
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org10November 2013 | Volume 8 | Issue 11 | e78065
Conceived and designed the experiments: RKL GH JM JPP. Performed
the experiments: RKL GH ARS. Analyzed the data: RKL GH ARS.
Wrote the paper: RKL ARS JM JPP.
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PLOS ONE | www.plosone.org12 November 2013 | Volume 8 | Issue 11 | e78065