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: firstname.lastname@example.org (JPP); email@example.com (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.
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org2 November 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.
1. Farese RV Jr, Walther TC (2009) Lipid droplets finally get a little R-E-S-P-E-C-
T. Cell 139: 855–860.
2. Martin S, Parton RG (2006) Lipid droplets: a unified view of a dynamic
organelle. Nat Rev Mol Cell Biol 7: 373–378.
3. Walther TC, Farese RV Jr (2009) The life of lipid droplets. Biochim Biophys
Acta 1791: 459–466.
4. Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants
and microorganisms. Prog Lipid Res 40: 325–438.
5. Bickel PE, Tansey JT, Welte MA (2009) PAT proteins, an ancient family of lipid
droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta 1791:
6. Brasaemle DL (2007) Thematic review series: adipocyte biology. The perilipin
family of structural lipid droplet proteins: stabilization of lipid droplets and
control of lipolysis. J Lipid Res 48: 2547–2559.
7. Walther TC, Farese RV Jr (2012) Lipid droplets and cellular lipid metabolism.
Annu Rev Biochem 81: 687–714.
8. Bostrom P, Rutberg M, Ericsson J, Holmdahl P, Andersson L, et al. (2005)
Cytosolic lipid droplets increase in size by microtubule-dependent complex
formation. Arterioscler Thromb Vasc Biol 25: 1945–1951.
9. Kunwar A, Tripathy S, Xu J, Mattson M, Anand P, et al. (2011) Mechanical
stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport.
Proc Natl Acad Sci USA 108: 18960–18965.
10. Targett-Adams P, Chambers D, Gledhill S, Hope RG, Coy JF, et al. (2003) Live
cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-
related protein. J Biol Chem 278: 15998–16007.
11. Welte MA, Gross SP, Postner M, Block SM, Wieschaus EF (1998)
Developmental regulation of vesicle transport in Drosophila embryos: forces
and kinetics. Cell 92: 547–557.
12. Gross S, Vershinin M, Shubeita G (2007) Cargo transport: two motors are
sometimes better than one. Curr Biol 17: R478–486.
13. Gross SP, Welte MA, Block SM, Wieschaus EF (2000) Dynein-mediated cargo
transport in vivo. A switch controls travel distance. J Cell Biol 148: 945–956.
14. Kulic I, Brown A, Kim H, Kural C, Blehm B, et al. (2008) The role of
microtubule movement in bidirectional organelle transport. Proc Natl Acad Sci
USA 105: 10011–10017.
15. Welte M (2004) Bidirectional transport along microtubules. Curr Biol 14: R525–
16. Kural C, Kim H, Syed S, Goshima G, Gelfand VI, et al. (2005) Kinesin and
Dynein Move a Peroxisome in Vivo: A Tug-of-War or Coordinated Movement?
Science 308: 1469–1472.
17. Lyn RK, Kennedy DC, Stolow A, Ridsdale A, Pezacki JP (2010) Dynamics of
lipid droplets induced by the hepatitis C virus core protein. Biochem Biophys
Res Commun 399: 518–542.
18. Muller MJ, Klumpp S, Lipowsky R (2008) Tug-of-war as a cooperative
mechanism for bidirectional cargo transport by molecular motors. Proc Natl
Acad Sci USA 105: 4609–4614.
19. Herker E, Ott M (2012) Emerging role of lipid droplets in host/pathogen
interactions. J Biol Chem 287: 2280–2287.
20. Pezacki JP, Singaravelu R, Lyn RK (2010) Host-virus interactions during
hepatitis C virus infection: a complex and dynamic molecular biosystem. Mol
Biosyst 6: 1131–1142.
21. Lavanchy D (2011) Evolving epidemiology of hepatitis C virus. Clin Microbiol
Infect 17: 107–115.
22. Tang H, Grise H (2009) Cellular and molecular biology of HCV infection and
hepatitis. Clin Sci 117: 49–65.
23. Alvisi G, Madan V, Bartenschlager R (2011) Hepatitis c virus and host cell lipids:
An intimate connection. RNA Biol 8: 258–269.
24. Herker E, Harris C, Hernandez Cl, Carpentier A, Kaehlcke K, et al. (2010)
Efficient hepatitis C virus particle formation requires diacylglycerol acyltransfer-
ase-1. Nat Med 16: 1295–1303.
25. Syed G, Amako Y, Siddiqui A (2010) Hepatitis C virus hijacks host lipid
metabolism. Trends Endocrinol Metab 21: 33–73.
26. Chisari F (2005) Unscrambling hepatitis C virus-host interactions. Nature 436:
27. Cle ´ment S, Negro F (2007) Hepatitis C virus: the viral way to fatty liver.
J Hepatol 46: 985–992.
28. McLauchlan J (2009) Lipid droplets and hepatitis C virus infection. Biochim
Biophys Acta 1791: 552–561.
29. Saka HA, Valdivia R (2012) Emerging Roles for Lipid Droplets in Immunity
and Host-Pathogen Interactions. Annu Rev Cell Dev Biol 28: 411–437.
30. Su A, Pezacki J, Wodicka L, Brideau A, Supekova L, et al. (2002) Genomic
analysis of the host response to hepatitis C virus infection. Proc Natl Acad Sci
USA 99: 15669–15743.
31. Jackel-Cram C, Babiuk LA, Liu Q (2007) Up-regulation of fatty acid synthase
promoter by hepatitis C virus core protein: genotype-3a core has a stronger
effect than genotype-1b core. J Hepatol 46: 999–1008.
32. Roingeard P, Depla M (2011) The birth and life of lipid droplets: learning from
the hepatitis C virus. Biol Cell 103: 223–231.
33. Moradpour D, Penin Fo, Rice C (2007) Replication of hepatitis C virus. Nat Rev
Microbiol 5: 453–463.
34. Barba G, Harper F, Harada T, Kohara M, Goulinet S, et al. (1997) Hepatitis C
virus core protein shows a cytoplasmic localization and associates to cellular lipid
storage droplets. Proc Natl Acad Sci USA 94: 1200–1205.
35. Boulant S, Douglas M, Moody L, Budkowska A, Targett-Adams P, et al. (2008)
Hepatitis C virus core protein induces lipid droplet redistribution in a
microtubule- and dynein-dependent manner. Traffic 9: 1268–1350.
36. Ait-Goughoulte M, Hourioux C, Patient R, Trassard S, Brand D, et al. (2006)
Core protein cleavage by signal peptide peptidase is required for hepatitis C
virus-like particle assembly. J Gen Virol 87: 855–915.
37. McLauchlan J, Lemberg MK, Hope G, Martoglio B (2002) Intramembrane
proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets.
The EMBO J 21: 3980–3988.
38. Okamoto K, Mori Y, Komoda Y, Okamoto T, Okochi M, et al. (2008)
Intramembrane processing by signal peptide peptidase regulates the membrane
localization of hepatitis C virus core protein and viral propagation. J Virol 82:
39. Perez-Berna AJ, Veiga AS, Castanho MA, Villalain J (2008) Hepatitis C virus
core protein binding to lipid membranes: the role of domains 1 and 2. J Viral
Hepat 15: 346–402.
40. Depla M, Uzbekov R, Hourioux C, Blanchard E, Le Gouge A, et al. (2010)
Ultrastructural and quantitative analysis of the lipid droplet clustering induced
by hepatitis C virus core protein. Cell Mol Life Sci 67: 3151–3161.
41. Kopp M, Murray C, Jones C, Rice C (2010) Genetic analysis of the carboxy-
terminal region of the hepatitis C virus core protein. J Virol 84: 1666–1739.
42. Targett-Adams P, Hope G, Boulant S, McLauchlan J (2008) Maturation of
hepatitis C virus core protein by signal peptide peptidase is required for virus
production. J Biol Chem 283: 16850–16859.
43. Alsaleh K, Delavalle P-Y, Pillez A, Duverlie G, Descamps V, et al. (2010)
Identification of basic amino acids at the N-terminal end of the core protein that
are crucial for hepatitis C virus infectivity. J Virol 84: 12515–12543.
44. Boulant S, Targett-Adams P, McLauchlan J (2007) Disrupting the association of
hepatitis C virus core protein with lipid droplets correlates with a loss in
production of infectious virus. J Gen Virol 88: 2204–2217.
45. Shavinskaya A, Boulant S, Penin F, McLauchlan J, Bartenschlager R (2007) The
lipid droplet binding domain of hepatitis C virus core protein is a major
determinant for efficient virus assembly. J Biol Chem 282: 37158–37169.
46. Jones D, McLauchlan J (2010) Hepatitis C virus: assembly and release of virus
particles. J Biol Chem 285: 22733–22742.
47. Lai C-K, Jeng K-S, Machida K, Lai M (2010) Hepatitis C virus egress and
release depend on endosomal trafficking of core protein. J Virol 84: 11590–
48. Wang C, Gale M Jr, Keller BC, Huang H, Brown MS, et al. (2005)
Identification of FBL2 as a geranylgeranylated cellular protein required for
hepatitis C virus RNA replication. Mol Cell 18: 425–434.
49. Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T, et al. (2007) The lipid
droplet is an important organelle for hepatitis C virus production. Nat Cell Biol
50. Evans CL, Potma EO, Puoris’haag M, Co ˆte ´ D, Lin CP, et al. (2005) Chemical
imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering
microscopy. Proc Natl Acad Sci USA 102: 16807–16812.
51. Pegoraro AF, Ridsdale A, Moffatt DJ, Jia Y, Pezacki JP, et al. (2009) Optimally
chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator.
Opt Express 17: 2984–2996.
52. Pegoraro AF, Ridsdale A, Moffatt DJ, Pezacki JP, Thomas B, et al. (2009) All-
fiber CARS microscopy of live cells. Opt Express 17: 20700–20706.
53. Pezacki JP, Blake JA, Danielson D, Kennedy DC, Lyn RK, et al. (2011)
Chemical contrast for imaging living systems: molecular vibrations drive CARS
microscopy. Nat Chem Biol 7: 137–182.
54. Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, et al. (2002) Expression of
hepatitis C virus proteins induces distinct membrane alterations including a
candidate viral replication complex. J Virol 76: 5974–5984.
55. Boulant S, Montserret R, Hope R, Ratinier M, Targett-Adams P, et al. (2006)
Structural determinants that target the hepatitis C virus core protein to lipid
droplets. J Biol Chem 281: 22236–22247.
56. Boulant S, Vanbelle C, Ebel C, Penin F, Lavergne J-P (2005) Hepatitis C Virus
Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein
Features. J Virol 79: 11353–11365.
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org 11 November 2013 | Volume 8 | Issue 11 | e78065
57. Burgess SA, Walker ML, Sakakibara H, Knight PJ, Oiwa K (2003) Dynein
structure and power stroke. Nature 421: 715–718.
58. Dodding M, Way M (2011) Coupling viruses to dynein and kinesin-1. EMBO J
59. Henry T, Gorvel J-P, Me ´resse S (2006) Molecular motors hijacking by
intracellular pathogens. Cell Microbiol 8: 23–32.
60. Lyman MG, Enquist LW (2009) Herpesvirus Interactions with the Host
Cytoskeleton. J Virol 83: 2058–2066.
61. McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, et al. (2002)
Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159:
62. Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, et al. (1999)
Microtubule-dependent plus- and minus end-directed motilities are competing
processes for nuclear targeting of adenovirus. J Cell Biol 144: 657–672.
63. Roohvand F, Maillard P, Lavergne JP, Boulant S, Walic M, et al. (2009)
Initiation of hepatitis C virus infection requires the dynamic microtubule
network: role of the viral nucleocapsid protein. J Biol Chem 284: 13778–13869.
64. Murray CL, Jones CT, Tassello J, Rice CM (2007) Alanine scanning of the
hepatitis C virus core protein reveals numerous residues essential for production
of infectious virus. J Virol 81: 10220–10231.
65. White SH, Wimley WC (1998) Hydrophobic interactions of peptides with
membrane interfaces. Biochim Biophys Acta 1376: 339–352.
66. Counihan N, Rawlinson S, Lindenbach B (2011) Trafficking of hepatitis C virus
core protein during virus particle assembly. PLoS Pathog 7: e1002302.
67. Shubeita GT, Tran SL, Xu J, Vershinin M, Cermelli S, et al. (2008)
Consequences of motor copy number on the intracellular transport of kinesin-
1-driven lipid droplets. Cell 135: 1098–1107.
68. Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF (2001)
Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-
mediated nuclear targeting of virus. EMBO J 20: 1310–1319.
69. Erhardt A, Hassan M, Heintges T, Ha ¨ussinger D (2002) Hepatitis C Virus Core
Protein Induces Cell Proliferation and Activates ERK, JNK, and p38 MAP
Kinases Together with the MAP Kinase Phosphatase MKP-1 in a HepG2 Tet-
Off Cell Line. Virology 292: 272–284.
70. Hayashi J, Aoki H, Kajino K, Moriyama M, Arakawa Y, et al. (2000) Hepatitis
C virus core protein activates the MAPK/ERK cascade synergistically with
tumor promoter TPA, but not with epidermal growth factor or transforming
growth factor a. Hepatology 32: 958–961.
71. Spaziani A, Alisi A, Sanna D, Balsano C (2006) Role of p38 MAPK and RNA-
dependent Protein Kinase (PKR) in Hepatitis C Virus Core-dependent Nuclear
Delocalization of Cyclin B1. J Biol Chem 281: 10983–10989.
72. Wileman T (2006) Aggresomes and Autophagy Generate Sites for Virus
Replication. Science 312: 875–878.
73. Wileman T (2007) Aggresomes and Pericentriolar Sites of Virus Assembly:
Cellular Defense or Viral Design? Annu Rev Microbiol 61: 149–167.
74. Neveu G, Barouch-Bentov R, Ziv-Av A, Gerber D, Jacob Y, et al. (2012)
Identification and Targeting of an Interaction between a Tyrosine Motif within
Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly. PLoS
Pathog 8: e1002845.
75. Harris C, Herker E, Farese RV, Ott M (2011) Hepatitis C Virus Core Protein
Decreases Lipid Droplet Turnover. J Biol Chem 286: 42615–42625.
76. Lyn RK, Kennedy DC, Sagan SM, Blais DR, Rouleau Y, et al. (2009) Direct
imaging of the disruption of hepatitis C virus replication complexes by inhibitors
of lipid metabolism. Virology 394: 130–172.
77. Mazumder N, Lyn RK, Singaravelu R, Ridsdale A, Moffatt DJ, et al. (2013)
Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain
2 of the Hepatitis C Virus Core Protein. PLoS One 8: e66738.
Lipid Droplet Velocity Changes by HCV Core-DII
PLOS ONE | www.plosone.org12 November 2013 | Volume 8 | Issue 11 | e78065