JOURNAL OF VIROLOGY, Oct. 2009, p. 10275–10279
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 19
Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel
Kimberly D. Erickson,1Robert L. Garcea,1* and Billy Tsai2*
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, 347 UCB, Boulder,
Colorado 80309,1and Department of Cell and Developmental Biology, University of Michigan Medical School,
109 Zina Pitcher Place, Room 3043, Ann Arbor, Michigan 481092
Received 13 May 2009/Accepted 9 July 2009
The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an
aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found
that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and
interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the
analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b
carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its
entry mechanism and pathophysiology.
Members of the polyomavirus (PyV) family, including sim-
ian virus 40 (SV40), murine PyV (mPyV), and BK virus
(BKV), bind cell surface gangliosides to initiate infection (2, 8,
11, 15). PyV capsids are assembled from 72 pentamers (cap-
someres) of the major coat protein VP1, with the internal
proteins VP2 and VP3 buried within the capsids (7, 12). The
VP1 pentamer makes direct contact with the carbohydrate
portion of the ganglioside (10, 12, 13) and dictates the speci-
ficity of virus interaction with the cell. Gangliosides are glyco-
lipids that contain a ceramide domain inserted into the plasma
membrane and a carbohydrate domain that directly binds the
virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15),
mPyV binds to gangliosides GD1a and GT1b (11, 15), and
BKV binds to gangliosides GD1b and GT1b (8).
Recently, a new human PyV designated Merkel cell PyV
(MCPyV) was identified in Merkel cell carcinomas, a rare but
aggressive skin cancer of neuroendocrine origin (3). It is as yet
unclear whether MCPyV is the causative agent of Merkel cell
carcinomas (17). A key to understanding the infectious and
transforming properties of MCPyV is the elucidation of its
cellular entry pathway. In this study, we identify a putative host
cell receptor for MCPyV.
Because an intact infectious MCPyV has not yet been iso-
lated, we generated recombinant MCPyV VP1 pentamers in
order to characterize cellular factors that bind to MCPyV. VP1
capsomeres have been previously shown to be equivalent to
virus with respect to hemagglutination properties (4, 16), and
the atomic structure of VP1 bound to sialyllactose has dem-
onstrated that the capsomere is sufficient for this interaction
(12, 13). The MCPyV VP1 protein (strain w162) was expressed
and purified as described previously (1, 6). Briefly, a glutathi-
one S-transferase-MCPyV VP1 fusion protein was expressed in
Escherichia coli and purified using glutathione-Sepharose af-
finity chromatography. The fusion protein was eluted using
glutathione and cleaved in solution with thrombin. The throm-
bin-cleaved sample was then rechromatographed on a second
glutathione-Sepharose column to remove glutathione trans-
ferase and any uncleaved protein. The unbound VP1 was then
chromatographed on a P-11 phosphocellulose column, and
peak fractions eluting between 400 and 450 mM NaCl were
collected. The purified protein was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fol-
lowed by Coomassie blue staining (Fig. 1A, left) and immuno-
blotting using an antibody (I58) that generally recognizes PyV
VP1 proteins (Fig. 1A, right) (9). Transmission electron mi-
croscopy (Philips CM10) analysis confirmed that the purified
recombinant MCPyV VP1 formed pentamers (capsomeres),
which did not assemble further into virus-like particles (Fig.
1B). In an initial screening of its cell binding properties, we
tested whether the MCPyV VP1 pentamers hemagglutinated
red blood cells (RBCs). The MCPyV VP1 pentamers were
incubated with sheep RBCs and assayed as previously de-
scribed (5). SV40 and mPyV recombinant VP1 pentamers
served as negative and positive controls, respectively. We
found that MCPyV VP1 hemagglutinated the RBCs with the
same efficiency as mPyV VP1 (protein concentration/hemag-
glutination unit) (Fig. 1C, compare rows B and C from wells 1
to 11), suggesting that MCPyV VP1 engages a plasma mem-
brane receptor on the RBCs. The recombinant murine VP1
protein used for comparison was from the RA strain, a small
plaque virus (4). Thus, MCPyV VP1 has the hemagglutination
characteristics of a small plaque mPyV (12, 13).
To characterize the chemical nature of the putative receptor
for MCPyV, total membranes from RBCs were purified as
described previously (15). The plasma membranes (30 ?g)
were incubated with MCPyV VP1 (0.5 ?g) and floated on a
discontinuous sucrose gradient (15). After fractionation, the
samples were analyzed by SDS-PAGE, followed by immuno-
blotting with I58. VP1 was found in the bottom of the gradient
* Corresponding author. Mailing address for R. L. Garcea: Depart-
ment of Molecular, Cellular, and Developmental Biology, University
of Colorado at Boulder, 347 UCB, Boulder, CO 80309. Phone: (303)
492-1669. Fax: (303) 492-1133. E-mail: email@example.com.
Mailing address for B. Tsai: Department of Cell and Developmental
Biology, University of Michigan Medical School, 109 Zina Pitcher
Place, Room 3043, Ann Arbor, MI 48109. Phone: (734) 764-4167. Fax:
(734) 764-5155. E-mail: firstname.lastname@example.org.
?Published ahead of print on 15 July 2009.
in the absence of the plasma membranes (Fig. 2A, first panel).
In the presence of plasma membranes, a fraction of the VP1
floated to the middle of the gradient (Fig. 2A, second panel),
supporting the hemagglutination results that suggested that
MCPyV VP1 binds to a receptor on the plasma membrane.
To determine whether the receptor is a protein or a lipid,
plasma membrane preparations (30 ?g) were incubated with
proteinase K (Sigma), followed by analysis with SDS-PAGE
and Coomassie blue staining. Under these conditions, the ma-
jority of the proteins in the plasma membranes were degraded
by the protease (Fig. 2B, compare lanes 1 and 2). Despite the
lack of proteins, the proteinase K-treated plasma membranes
bound MCPyV VP1 as efficiently as control plasma mem-
branes (Fig. 2A, compare the second and third panels), dem-
onstrating that MCPyV VP1 interacts with a protease-resistant
receptor. The absence of VP1 in the bottom fraction in Fig. 2A
(third panel) is consistent with the fact that the buoyant density
of the membranes is lowered by proteolysis. Of note, a similar
result was seen with binding of the mPyV to the plasma mem-
brane (15). Binding of MCPyV to the cell surface of two
human tissue culture cells (i.e., HeLa and 293T) was also
largely unaffected by pretreatment of the cells with proteinase
K (Fig. 2C and D, compare lanes 1 and 2), further indicating
that a nonproteinaceous molecule on the plasma membrane
engages the virus.
We next determined whether the protease-resistant receptor
contains a sialic acid modification. Plasma membranes (10 ?g)
were incubated with a neuraminidase (?2-3,6,8 neuraminidase;
Calbiochem) to remove the sialic acid groups. In contrast to
the control plasma membranes, the neuraminidase-treated
membranes did not bind MCPyV VP1 (Fig. 2E, compare first
and second panels), indicating that the MCPyV receptor in-
cludes a sialic acid modification.
Gangliosides are lipids that contain sialic acid modifications.
We asked if MCPyV VP1 binds to gangliosides similar to other
PyV family members. The structures of the gangliosides used
in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b)
are depicted in Fig. 3A. To assess a possible ganglioside-VP1
interaction, we employed a liposome flotation assay estab-
lished previously (15). When liposomes (consisting of phos-
phatidyl-choline [19 ?l of 10 mg/ml], -ethanolamine [5 ?l of 10
mg/ml], -serine [1 ?l of 10 mg/ml], and -inositol [3 ?l of 10
mg/ml]) were incubated with MCPyV VP1 and subjected to the
sucrose flotation assay, the VP1 remained in the bottom frac-
tion (Fig. 3B, first panel), indicating that VP1 does not interact
with these phospholipids. However, when liposomes contain-
ing GT1b (1 ?l of 1 mM), but not GM1 (1 ?l of 1 mM) or
GD1a (1 ?l of 1 mM), were incubated with MCPyV VP1, the
vesicles bound this VP1 (Fig. 3B). A low level of virus floated
partially when incubated with liposomes containing GD1b
FIG. 1. Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie
blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated.
(B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 ?g/ml and absorbed onto Formvar/
carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission
electron microscopy at 80 kV. Bar ? 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40
VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 ?g/ml (well
1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 ?g/ml
(Fig. 3B), perhaps reflecting a weak affinity between MCPyV
and GD1b. Importantly, MCPyV binds less efficiently to neura-
minidase-treated GT1b-containing liposomes than to GT1b-
containing liposomes (Fig. 3B, sixth panel), suggesting that the
GT1b sialic acids are involved in virus binding. This finding is
consistent with the ability of neuraminidase to block MCPyV
binding to the plasma membrane (Fig. 2E). The level of virus
flotation observed in the neuraminidase-treated GT1b-con-
taining liposomes is likely due to the inefficiency of the neur-
aminidase reaction with a high concentration of GT1b used to
prepare the vesicles.
As controls, GM1-containing liposomes bound SV40 (Fig.
3C), GD1a-containing liposomes bound mPyV (Fig. 3D), and
GD1b-containing liposomes bound BKV (Fig. 3E), demon-
strating that the liposomes were functionally intact. We note
that, while all of the MCPyV VP1 floated when incubated with
liposomes containing GT1b (Fig. 3B, sixth panel), a significant
fraction of SV40, mPyV, and BKV VP1 remained in the bot-
tom fraction despite being incubated with liposomes contain-
ing their respective ganglioside receptors (Fig. 3C to E, second
panels). This result is likely due to the fact that in contrast to
MCPyV, which are assembled as pentamers (Fig. 1B), the
SV40, mPyV, and BKV used in these experiments are fully
assembled particles: their larger and denser nature prevents
efficient flotation. Nonetheless, we conclude that MCPyV VP1
binds to ganglioside GT1b efficiently.
The observation that GD1a does not bind to MCPyV VP1
suggests that the monosialic acid modification on the right
branch of GT1b (Fig. 3A) is insufficient for binding. Similarly,
the failure of GD1b to bind MCPyV VP1 suggests that the
sialic acid on the left arm of GT1b is necessary for binding.
Together, these observations suggest that MCPyV VP1 inter-
acts with sialic acids on both branches of GT1b (Fig. 4). A
recent structure of SV40 VP1 in complex with the sugar por-
tion of GM1 (10) demonstrated that although SV40 VP1 binds
both branches of GM1 (Fig. 4), only a single sialic acid in GM1
is involved in this interaction. In the case of mPyV, structures
of mPyV VP1 in complex with different carbohydrates (12, 13)
revealed that the sialic acid-galactose moiety on the left branch
of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig.
FIG. 2. MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV
VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the
fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control
and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with
proteinase K (4 ?g/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As
described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with
or without ?2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.
VOL. 83, 2009 NOTES 10277
FIG. 3. MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic
linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides.
The samples were analyzed as described in the legend to Fig. 2A. Where indicated, GT1b-containing liposomes were pretreated with ?2-3,6,8
neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as
described in the legend to panel B.
10278 NOTESJ. VIROL.
4). Although no structure of BKV in complex with the sugar Download full-text
portion of GD1b (or GT1b) is available, in vitro binding stud-
ies (8) have suggested that the disialic acid modification on the
right branch of GD1b (and GT1b) is responsible for binding
BKV VP1 (Fig. 4). Thus, it appears that the unique feature of
the MCPyV VP1-GT1b interaction is that the sialic acids on
both branches of this ganglioside are likely involved in capsid
The identification of a potential cellular receptor for
MCPyV will facilitate the study of its entry mechanism. An
important issue for further study is to determine whether
MCPyV targets Merkel cells preferentially, and if so, whether
GT1b is found in higher levels in these cells to increase sus-
We acknowledge support from NIH grants AI064296 (to B.T.) and
CA37667 (to R.L.G.).
1. Bird, G., M. O’Donnell, J. Moroianu, and R. L. Garcea. 2008. Possible role
for cellular karyopherins in regulating polyomavirus and papillomavirus cap-
sid assembly. J. Virol. 82:9848–9857.
2. Campanero-Rhodes, M. A., A. Smith, W. Chai, S. Sonnino, L. Mauri, R. A.
Childs, Y. Zhang, H. Ewers, A. Helenius, A. Imberty, and T. Feizi. 2007.
N-glycolyl GM1 ganglioside as a receptor for simian virus 40. J. Virol.
3. Feng, H., M. Shuda, Y. Chang, and P. S. Moore. 2008. Clonal integration of
a polyomavirus in human Merkel cell carcinoma. Science 319:1096–1100.
4. Freund, R., R. L. Garcea, R. Sahli, and T. L. Benjamin. 1991. A single amino
acid substitution in polyomavirus VP1 correlates with plaque size and hem-
agglutination behavior. J. Virol. 65:350–355.
5. Garcea, R. L., and T. L. Benjamin. 1983. Isolation and characterization of
polyoma nucleoprotein complexes. Virology 130:65–75.
6. Kean, J. M., S. Rao, M. Wang, and R. L. Garcea. 2009. Seroepidemiology of
human polyomaviruses. PLoS Pathog. 5(3):e1000363.
7. Liddington, R. C., Y. Yan, J. Moulai, R. Sahli, T. L. Benjamin, and S. C.
Harrison. 1991. Structure of simian virus 40 at 3.8-A resolution. Nature
8. Low, J. A., B. Magnuson, B. Tsai, and M. J. Imperiale. 2006. Identification
of gangliosides GD1b and GT1b as receptors for BK virus. J. Virol. 80:1361–
9. Montross, L., S. Watkins, R. B. Moreland, H. Mamon, D. L. D. Caspar, and
R. L. Garcea. 1991. Nuclear assembly of polyomavirus capsids in insect cells
expressing the major capsid protein VP1. J. Virol. 65:4991–4998.
10. Neu, U., K. Woellner, G. Gauglitz, and T. Stehle. 2008. Structural basis of
GM1 ganglioside recognition by simian virus 40. Proc. Natl. Acad. Sci. USA
11. Smith, A. E., H. Lilie, and A. Helenius. 2003. Ganglioside-dependent cell
attachment and endocytosis of murine polyomavirus-like particles. FEBS
12. Stehle, T., Y. Yan, T. L. Benjamin, and S. C. Harrison. 1994. Structure of
murine polyomavirus complexed with an oligosaccharide receptor fragment.
13. Stehle, T., and S. C. Harrison. 1996. Crystal structures of murine polyoma-
virus in complex with straight-chain and branched-chain sialyloligosaccha-
ride receptor fragments. Structure 4:183–194.
14. Reference deleted.
15. Tsai, B., J. M. Gilbert, T. Stehle, W. Lencer, T. L. Benjamin, and T. A.
Rapoport. 2003. Gangliosides are receptors for murine polyoma virus and
SV40. EMBO J. 22:4346–4355.
16. Zullo, J. N., C. D. Stiles, and R. L. Garcea. 1987. Induction of c-myc and c-fos
by polyomavirus: distinct roles for the capsid protein VP1 and the viral early
proteins. Proc. Natl. Acad. Sci. USA. 84:1210–1214.
17. zur Hausen, H. 2008. Novel human polyomaviruses—re-emergence of a well
known virus family as possible human carcinogens. Int. J. Cancer 123:247–
FIG. 4. A potential model of the different VP1-ganglioside interactions (see the text for discussion).
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