Chaipan et al. Retrovirology 2010, 7:47
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Open Access

RESEARCH

Incorporation of podoplanin into HIV released
from HEK-293T cells, but not PBMC, is required for
efficient binding to the attachment factor CLEC-2
Research

Chawaree Chaipan†1,2, Imke Steffen†3, Theodros Solomon Tsegaye†3, Stephanie Bertram3, Ilona Glowacka3,
Yukinari Kato4, Jan Schmökel5, Jan Münch5, Graham Simmons6, Rita Gerardy-Schahn7 and Stefan Pöhlmann*1,2,3

Abstract
Background: Platelets are associated with HIV in the blood of infected individuals and might modulate viraldissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and thenovel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells.
Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission.
Results: Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize theviral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed thatthe cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells andincorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed thatvirion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines andplatelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral bloodmononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander
cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon
induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin
expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to Tcells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand.
Conclusions: Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while
incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived
viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding thatdeserves further investigation.
BackgroundThe envelope protein (Env) of the human immunodeficiency virus (HIV), a heavily glycosylated type I transmembrane protein, mediates infectious viral entry into
target cells [1]. This process depends on the interactionsof Env with proteins displayed at the surface of host cells.
All primary HIV-1 isolates characterized to date engage
the CD4 protein as receptor for infectious entry [2,3].
Upon binding to CD4, a coreceptor binding site is gener* Correspondence: poehlmann.stefan@mh-hannover.de
1 Nikolaus-Fiebiger-Center for Molecular Medicine, University Hospital

Erlangen, 91054 Erlangen, Germany† Contributed equallyated or exposed in Env, which allows engagement of thechemokine coreceptors CCR5 and CXCR4. The interactions of Env with CD4 and coreceptor are essential for
infectious entry, and the interacting surfaces are key targets for preventive and therapeutic approaches [2,3]. For
instance, a small molecule inhibitor of Env binding to
CCR5, maraviroc, blocks spread of CCR5-tropic HIV andis used as salvage therapy for patients who do notrespond to conventional HIV therapy [4,5].
Receptor expression levels can limit HIV entry into
host cells [6,7], and this limitation can be overcome byconcentrating virions onto target cells by, for example,
centrifugation or polybrene treatment [8]. A constantly

Full list of author information is available at the end of the article© 2010 Chaipan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons

BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

Chaipan et al. Retrovirology 2010, 7:47
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accumulating body of evidence suggests that certain hostcell factors can also promote viral attachment to cells andcan thereby increase infection efficiency [9,10]. A striking
example is the interaction of HIV with a semen-derived
fragment of prostatic acidic phosphatase, termed SEVI
(for Semen Enhancer of Virus Infection) [11]. SEVI, anamyloidogenic peptide, forms fibrils in human semen
which capture HIV and concentrate virions onto target
cells [11]. As a consequence, SEVI boosts viral infectivity
and might increase the risk of acquiring HIV infection
upon sexual intercourse. Incorporation of host cell factors into the HIV envelope can also increase viral infectivity. The augmentation of infectivity is due to theinteraction of the virion-incorporated factors with theircognate receptors on HIV target cells, as exemplified by
the up to 100-fold increased infectivity of ICAM-1-bearing viruses for LFA-1 positive target cells [12,13]. Finally,
attachment of HIV to dendritic cells can also promote
HIV infection of adjacent T-cells [14,15], and this property has been associated with the expression of DC-SIGN[16], a calcium-dependent (C-type) lectin which recognizes mannose-rich carbohydrates on the HIV Env protein [17-19]. Engineered expression of DC-SIGN on
certain cell lines promotes receptor-dependent infectionof these cells (termed infection in cis) [20] or of adjacenttarget cells (termed infection in trans, or transmission)[16], and it has been suggested that DC-SIGN might promote HIV spread in and between individuals [16]. However, this hypothesis is intensely debated [21-25]. In fact,
several lines of evidence suggest that DC-SIGN mightmainly function as a pathogen recognition receptor,
which promotes HIV uptake for MHC presentation andthereby exerts a protective function against HIV infection[23-27].
We and others have previously shown that apart fromdendritic cells, platelets also express DC-SIGN and thatthese cell fragments bind to HIV in a mainly DC-SIGNdependent manner [28,29]. However, the HIV binding
activity of platelets could be partially inhibited by antisera
specific for the newly identified HIV attachment factor
CLEC-2 [29], indicating that CLEC-2 contributes to HIV
capture by platelets. CLEC-2 is a lectin-like protein, andits putative carbohydrate recognition sequence contains
17 amino acid residues highly conserved between C-type
lectins [30]. Binding of the snake venom toxin rhodocytin
to CLEC-2 triggers Syk-dependent signalling in platelets
which causes platelet degranulation [31,32]. Residues in
CLEC-2 which are required for binding to rhodocytinhave been defined [33,34]. However, it is at present
unclear how CLEC-2 interacts with HIV.
Here, we report that CLEC-2, unlike DC-SIGN, does
not bind to the viral Env protein, but to a cellular factor
incorporated into the viral envelope. For viruses produced in the kidney-derived cell line 293T, this factor wasPage 2 of 18

found to be podoplanin (also termed aggrus), a cellularmucin-like glycoprotein expressed by kidney podocytes(which are known to be susceptible to HIV infection [35])and lymphatic endothelium [36-38]. Podoplanin expression was not detected on viable, but on apoptotic T-cells
and on apoptotic peripheral blood mononuclear cells(PBMCs). However, apoptosis of HIV infected T-cells was
not associated with podoplanin expression. Nevertheless,
CLEC-2 mediated trans-infection of HIV generated in
PBMCs, indicating that these cells might express a so far
unidentified CLEC-2-ligand which can facilitate CLEC-2dependent HIV capture.

Methods
Cell culture and transfection

293T, 293 T-REx [19], GP2 293 (Clontech, California,
USA) and CHO cells were maintained in Dulbecco's
modified Eagle medium (DMEM) supplemented with
10% fetal calf serum (FCS, Biochrom, Germany), penicillin and streptomycin. In addition, blasticidin and zeocin
were used for selection of 293 T-REx cells expressing
CLEC-2 upon induction with doxycycline (Sigma, Germany). CHO Lec1 and CHO Lec2 cells [39-41] were cultured in αMEM (PAA, Germany), supplemented with
10% FCS and antibiotics. B-THP, B-THP DC-SIGN, BTHP CLEC-2 (Raji B cells that were engineered to express
DC-SIGN [42], CLEC-2 [29] or empty vector), C8166SEAP cells [43] and CEM×174 5.25 M7 (abbreviatedCEM×174 R5) cells [44], the latter expressing exogenous
CCR5, were cultured in RPMI 1640 medium (PAA, Germany) in the presence of antibiotics and 10% FCS. All
cells were cultured at 37°C and 5% CO2. Highly purified
platelets were obtained from the "Transfusionsmedizinische und Hämostaseologische Abteilung" of the University
Hospital Erlangen. Alternatively, platelets were preparedfrom whole blood by centrifugation at 1200 rpm at RT.
The upper platelet-rich plasma was collected and centrifuged at 4000 rpm for 20 min at RT. Subsequently, thesupernatant was removed, and platelets were resuspended in RPMI 1640 medium supplemented with 10%
FCS and antibiotics. PBMCs were isolated from whole
blood or leukocyte filters by centrifugation through a
Ficoll gradient and either cultured in RMPI 1640 medium
supplemented with 10% FCS and antibiotics or stimulated with PHA (Sigma) at a concentration of 5 μg/ml andIL-2 (Roche) at a concentration of 10 U/ml.
Plasmids

The NL4-3-based reporter virus bearing EGFP in place ofnef was generated by splice overlap extension (SOE) PCR.
Briefly, a NL4-3 env fragment was amplified using oligonucleotides pJM206 (binding upstream of the singular
HpaI restriction site in env), and pJM394 (binding to the
3' end of env and also containing the first three triplets ofChaipan et al. Retrovirology 2010, 7:47
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EGFP) and pBRNL4-3 [45] as template. EGFP was amplified from pEGFP-C1 (Clontech) using primers JM395(binding to EGFP start sequences) and JM396 (introducing a MluI site downstream of the EGFP stop codon).
Both PCR fragments were fused by SOE PCR using primers pJM206 and pJM396. The resulting env-EGFP fragment was cloned via HpaI and MluI into pBRNL4-3_nef+
Δ1Δ2 [46] resulting in the generation of pBRNL4-3-EGFP
in which nef was replaced by EGFP. Oligonucleotide
sequences (env sequences in bold; EGFP sequences initalics, MluI restriction site underlined): pJM206 5'TGGAACTTCTGGGACGCAGG-3'; pJM394 5'-GCTCACCAT CTTATAGCAAAATCC;JM395 GCTATAAGATGGTGAGCAAGGGCG-3';JM396 5'-CGTACGCG
TTACTTGTACAGC-3'. The gp120-Fc-IgG1 construct[47] was generated by amplifying a codon-optimized
gp120 (JRFL) [48] with primers gp120_BamHI 5'-GAGTGGATCCCTTATCGTCGTCATCCTTGTAATCC-3'(sense) and gp120_HindIII 5'-GTACGAAGCTTGTGGAGAAGCTGTGGGTGAC-3' (antisense), followed byinsertion of the PCR fragment in the BamHI and HindIII
restriction sites of the Fc-IgG1 encoding plasmid pAB61[49]. For generating the CLEC-2-Fc-IgG1 fusion construct, sense primer 5'-GTACGAAGCTTTGCAGCCCCTGTGACACAAAC-3'and antisense primer 5'-GAGTGGATCCAGGTAGTTTCCACCTTGG-3' were used for
PCR amplification, and the product was cloned into
pAB61 using the HindIII and BamHI restriction sites.
CLEC-2 mutants bearing single amino acid changes weregenerated by overlap extension PCR. The oligonucleotides 5'-GCCGGATCCACCATGCAGGATGAAGATGGATACATC-3' (sense) and GCCGAATTCTTAAGGTA
GTTGGTCCACCTTGG (antisense) were used as outerprimers and combined with the following inner primers:5'-GATGGAAAAGGAGCCATGAATTGTGC-3'(sense) and 5'-AGCACAATTCATGGCTCCTTTTCCAT-3' (antisense) for generation of mutant CLEC-2
N192A, 5'-TTGAGTTTTTGGCCGATGGAAAAGG-3'(sense) and 5'-TCCTTTTCCATCGGCCAAAAACTCA3' (antisense) for mutant CLEC-2 E187A, 5'-GTTTTTGGAAGATGGAGCCGGAAATATGAATTGTG-3' (sense)and 5'-AATTCATATTTCCGGCTCCATCTTCCAAAA3' (antisense) for mutant CLEC-2 K190A, 5'-GCAACATTG TGGAATATATTGCGGCGCGCACCCATCTGATTC-3' (sense) and 5'-GCGCCGCAATATATTCCACAATG-3' (antisense) for mutant CLEC-2 K150A.
For generation of DC-SIGN-Fc-IgG1, primers 5'-GTACGAAGCTTGAACGCCTGTGCCACCCCTG-3' (sense)
and5'-GAGTGGATCCCGCAGGAGGGGGGTTTGGGG-3' (antisense) were used. The resulting PCR fragment was cloned into pAB61, using the HindIII and
BamHI restriction sites. A PCR fragment encoding theextracellular domain of podoplanin fused to the Fc por-Page 3 of 18

tion of human immunoglobulin was generated as
described above, employing primers 5'-GCCAAGCTTGCCAGCACAGGCCAGCCAGAAGATG-3' (sense) and
5'-GCGGGATCCTGTTGACAAACCATCTTTCT CAA
C-3' (antisense) and inserted into the pAB61 plasmid via
the HindIII and BamHI restriction sites (italics). The
identity of all PCR amplified sequences was confirmed bysequencing with an ABI3700 genetic analyzer (Applied
Biosystems) according to the manufacturer's instructions.
The plasmid used for transient expression of podoplanin(podoplanin in pcDNA3) has been previously described[38].
Viruses and transmission analyses

Replication-competent HIV-1 NL4-3, NL4-3 luc [50] andNL4-3 EGFP were generated as described elsewhere [50].
Briefly, 293T cells were transfected with plasmids encoding proviral DNA, and culture medium was changed 12 hpost transfection. Culture supernatants were harvested at
48 h post transfection and filtered through a 0.45 μm filter, aliquoted and stored at -80°C. Transmission analyses
were carried out as described [29]. Briefly, B-THP control
cells, B-THP-DC-SIGN and B-THP-CLEC-2 cells [29,42]or platelets were incubated with virus for 3 h at 37°C, andunbound virus was removed by washing with fresh culture medium. Cells were then incubated with CEM×174
R5 target cells and luciferase activities in cellular lysates
were determined three days after the start of the cocultivation by employing a commercially available system(Promega, Germany).
Binding studies with soluble proteins

For generating soluble Zaire Ebolavirus glycoprotein(ZEBOV-GP)-Fc- [51], DC-SIGN-Fc-, CLEC-2-Fc- andPodoplanin-Fc-fusion proteins, 293T cells were calcium
phosphate-transfected with the respective plasmids orpAB61 control plasmid encoding only the Fc-portion of
IgG1. For transfection of CHO and mutant cell lines,
Lipofectamine 2000 transfection reagent (Invitrogen,Germany) was used according to the manufacturer's protocol. The cells were washed with PBS and the culture
medium was replaced by FCS-free medium at 12 h posttransfection and supernatants were harvested 48 h posttransfection. Subsequently, supernatants were concentrated using Centricon Plus-20 size-exclusion centrifugal
filters (Millipore, Germany; centrifugation at 4000 g for
15 minutes), aliquoted, and stored at -80°C. To employ
comparable amounts of soluble proteins for binding studies, Fc-fusion protein preparations were normalized byWestern blot, employing an anti-human IgG-horseradish
peroxidase conjugate for detection (Dianova, Germany).
To assess binding, 5 × 105 cells were incubated with Fcfusion proteins and Fc-control protein at 4°C for 45 minutes. Subsequently, the cells were washed with FACS buf-

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fer and stained with Cy5-conjugated anti-human IgG
secondary antibody for 30 minutes at 4°C. Cell-staining
was then analyzed by flow cytometry, employing a
Cytomics FC500 flow cytometer (Beckman-Coulter, Florida, USA), and data were analyzed with FCS Express
FACS analysis software (De Novo Software, Los Angeles,
USA).
Analysis of podoplanin surface expression

Analyses of podoplanin surface expression were performed by flow cytometry, using the podoplanin specificantibodies NZ-1 or 18H5 (Acris, Germany) in combination with secondary anti rat/mouse antibody coupled to
Cy5 (Dianova, Germany). Cells were incubated with 10
μg/ml antibody in PBS supplemented with 5% FCS for 30
minutes at 4°C. Subsequently, PBS supplemented with 5%
FCS was added, and the cells were pelleted by centrifugation (1200 rpm, 4°C for 5 minutes). Finally, cells were
resuspended in fixans (1.5% paraformaldehyde) and incubated for 30 minutes at 4°C before staining was analyzed
by flow cytometry. For all measurements 20,000 gatedevents were collected.
Knock-down of podoplanin expression by shRNA

For stable knock-down of podoplanin in 293T cells, shRNAs were constructed by using shRNA Hairpin Oligonucleotide Sequence Designer Tool (Clontech, California,
USA). The podoplanin specific shRNA 137 contained thetarget shRNA sequence, a hairpin loop region "TTCAAGAGA" and an antisense shRNA sequence followed by apol III terminator sequence. The shRNA was constructedby annealing shRNA137sense_BamHI: 5'GATCCGCGAAGATGATGTGGTGACTTTCAAGAGAAGTCACC ACATCATCTTCGTTTTTTACGCGTG3' andshRNA137antisense_EcoRI: 5'AATTCACGCGTAAAAAACGAAGATGATGTGGTGACTTCTCTTGAAAGTCA
CCACATCATCTTCGCG3' followed by insertion of the
double stranded fragment into the retroviral vector pSIREN-IRES-EGFP-RetroQ [52], using restriction enzymes
BamHI and EcoRI, respectively. This vector allows stable
expression of small hairpin RNAs in transduced cells,
which can be readily identified and selected due to vector
encoded genes for puromycin resistance and EGFP
(enhanced green fluorescence protein) expression. Retroviral transduction was performed by transient expressionof the shRNA constructs and VSV-G in the packaging cellline GP2-293 (Clontech, California, USA). At 48 h posttransfection, cell supernatants were harvested, andviruses were concentrated by ultracentrifugation for 2 h
at 4°C. Pelleted virions were resuspended in 2 ml medium
containing 2 μg/ml polybrene (Sigma-Aldrich, Germany)
and were used for transduction of 1 × 106 293T cells. At
24 h post transduction, cells were washed and incubatedfor 3 days. Subsequently, transduced cells were selected inPage 4 of 18

medium containing 10 μg/ml puromycin (Sigma-Aldrich,Germany).
Apoptosis induction

For apoptosis induction cells were incubated with 1 μM
staurosporine (New England Biolabs, Germany), 25 μg/
ml cycloheximide (Sigma-Aldrich, Germany) or 0.1%
DMSO as a control in culture medium for 14 h unlessotherwise stated. Cells were stained for apoptosis with
PE-conjugated annexin V (R&D Systems, Minnesota,
USA) and for necrosis with 7-aminoactinomycin D (7AAD, Sigma, Germany). Specifically, cells were incubatedwith 5 μl annexin V or 7-AAD for 20 min at room temperature and then washed with PBS supplemented with
5% FCS. Subsequently, cells were fixed in 1.5% paraformaldehyde for 30 minutes at 4°C. Staining was analyzed
within 30 minutes after completion of fixation by flow
cytometry. For all measurements 20,000 gated eventswere collected.
Inhibition of antibody binding by soluble podoplanin

The podoplanin specific antibodies 18H5 and NZ-1
(Acris, Germany) were pre-incubated with concentrated,soluble podoplanin-Fc fusion protein for 30 minutes at
4°C before staining of apoptotic cells for subsequent
FACS analysis.
Statistical analyses

Statistical significance was determined by employing a
two-tailed student's t-test for paired samples.

Results
Efficient binding of soluble CLEC-2 to 293T cells does not
require expression of the HIV-1 envelope protein

In order to better understand HIV-1 interactions with
CLEC-2, we first asked if CLEC-2, like DC-SIGN [16],binds to the HIV-1 envelope protein (Env). For this, we
generated soluble versions of DC-SIGN and CLEC-2 byfusing the extracellular domain of these lectins to the Fcportion of human immunoglobulin. Soluble DC-SIGN
bound to control transfected 293T cells with higher efficiency than the Fc-control protein (Fig. 1A), most likely
due to recognition of cellular proteins harbouring highmannose and/or fucose containing glycans, which arebound by DC-SIGN [17-19]. Notably, however, binding
was substantially enhanced upon expression of the HIV-1
NL4-3 Env protein on 293T cells (Fig. 1A), indicating that
DC-SIGN binds to HIV-1 Env, as expected from published data [16]. Finally, the interaction of soluble DCSIGN with control cells and Env expressing cells was specific, since binding could be inhibited by the mannosepolymer mannan, a previously described inhibitor of DCSIGN interactions with ligands [16]. Soluble CLEC-2 also
bound to 293T cells with higher efficiency than the Fccontrol protein (Fig. 1A). However, in stark contrast toChaipan et al. Retrovirology 2010, 7:47
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Page 5 of 18

A)
Soluble DC-SIGN

1000

Soluble CLEC-2

p = 0.068

ControlB)

60

HIV-1 Env

Control

CLEC-2-Fc+ Mannan

CLEC-2-Fc

CLEC-2-Fc+ Mannan

CLEC-2-Fc

Fc-Control

DC-SIGN-Fc+ Mannan

DC-SIGN-Fc

DC-SIGN-Fc+ Mannan

10

DC-SIGN-Fc

100

Fc-Control

GMCF

p = 0.059

HIV-1 Env

p = 0.09

50

Fc-Control

GMCF

40

gp120-Fc

30
20
10
0

Control CLEC-2 DC-SIGN

Figure 1 CLEC-2 does not recognize the viral Env protein. (A) 293T cells were either control transfected with empty vector or transfected with an
HIV-1 NL4-3 Env expression plasmid. Subsequently, the cells were preincubated with PBS or mannan and then DC-SIGN-Fc (left panel) or CLEC-2-Fc(right panel) fusion proteins or an Fc-control protein (black bars) were added. Unbound proteins were removed by washing and bound proteins detected by flow cytometry. The results represent the average of the geometric mean channel fluorescence (GMCF) measured in four independent experiments. Error bars indicate standard error of the mean (SEM). (B) 293T cells were transfected with DC-SIGN, CLEC-2 or empty vector and incubatedwith soluble HIV-1 Env gp120-Fc fusion protein or control Fc-protein. Unbound proteins were removed by washing and bound proteins detected byflow cytometry. The results represent the average ± SEM of the GMCF measured in three independent experiments. GMCF: geometric mean channel
fluorescence, SEM: standard error of the mean.

Chaipan et al. Retrovirology 2010, 7:47
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the results obtained with soluble DC-SIGN, the interaction was not inhibited by mannan and was not enhancedby expression of the viral Env protein. In agreement withthese results, soluble HIV-1 Env protein bound specifically to DC-SIGN but not to CLEC-2 expressing cells (Fig.
1B). We therefore concluded that CLEC-2, in contrast to
DC-SIGN, does not capture HIV-1 Env. Instead, CLEC-2
seemed to recognize a cellular factor expressed on 293T
cells, and binding to this factor did not depend on recognition of high-mannose carbohydrates.
Podoplanin, a recently identified CLEC-2 ligand, isexpressed on 293T cells

The cellular mucin podoplanin was recently shown tointeract with CLEC-2 [53]. Podoplanin is endogenously
expressed by kidney podocytes [37]. Therefore, we investigated if the kidney-derived cell line 293T also expresses
podoplanin. Flow cytometric analysis indeed revealed
high levels of podoplanin on the surface of 293T cells(Fig. 2A). Expression was further enhanced upon transfection of 293T cells with a podoplanin expression plasmid (Fig. 2A), and higher levels of podoplanin resulted in
more efficient binding of soluble CLEC-2 (Fig. 2B). In
contrast, no binding to the lymphoid cell line CEM×175
R5 was detected (Fig. 2B), which was podoplanin negative(see below). We then used soluble podoplanin to confirm
the interaction with CLEC-2. For this, CLEC-2 expression was induced on 293 T-REx CLEC-2 cells, and binding of soluble podoplanin fused to the Fc-portion ofhuman immunoglobulin was analyzed by flow cytometry.
Efficient binding of soluble podoplanin was observed
only upon induced expression of CLEC-2, and a control
Fc protein did not bind to the CLEC-2 expressing cells(Fig. 2C and data not shown). Thus, 293T cells, which weand many others frequently use for production of HIV-1
stocks, express podoplanin; and podoplanin specificallyinteracts with CLEC-2.
Glycosylation of podoplanin is required for efficientbinding to CLEC-2

We next sought to elucidate the determinants governing
efficient interactions between podoplanin and CLEC-2.
For instance, it is at present unclear if glycosylation of
podoplanin is required for binding to CLEC-2. Watson
and colleagues demonstrated that binding of CLEC-2 tothe snake venom protein rhodocytin is glycosylation
independent, and defined several amino acids in CLEC-2
which contributed to efficient rhodocytin binding[33,34]. Thus, mutations K150A, E187A, K190A andN192A decreased binding of CLEC-2 to rhodocytin in
surface plasmon resonance binding studies [34]. We
addressed if these residues were also required for binding
to soluble podoplanin. Flow cytometric analysis showedthat all changes, with the exception of K190A were com-

Page 6 of 18

patible with efficient expression of CLEC-2 (Fig. 3A).
Wild type CLEC-2 and all mutants, except K190A, boundto soluble podoplanin with similar efficiency, indicatingthat the CLEC-2 residues involved in rhodocytin binding
were not important for binding to podoplanin. Podoplanin contains sialylated O-glycans [54], and we next analyzed if glycosylation of podoplanin is essential forbinding to CLEC-2. For this, podoplanin-Fc fusion proteins were produced in wt CHO cells or CHO cells that
due to defects in either the medial Golgi localized Nacetylglucosaminyltransferase I (CHO Lec1) or the transGolgi localized CMP-sialic acid transporter (CHO Lec2)
have lost their abilities to produce complex N-glycans andsialylated glycoconjugates, respectively [39-41]. Soluble
proteins were concentrated from cellular supernatants bysize-exclusion filtration, and Western blot analysis
showed that the podoplanin-Fc preparations containedroughly comparable amounts of protein (Fig. 3B), while
the Fc-control protein preparation was more concentrated. When binding to CLEC-2 was analyzed in a
FACS-based assay, podoplanin produced in Lec1 cells
still bound to CLEC-2 with appreciable efficiency (Fig.
3C). In contrast, podoplanin produced in Lec2 cells andthus almost completely lacking sialoglycoconjugates did
not show significant binding to CLEC-2 (Fig. 3C). The
observed differences indicate that the presence of sialic
acid is essential for binding to CLEC-2. Moreover,because N-glycans are exclusively of the high-mannose
type if proteins are expressed in Lec1 cells, this finding
provides evidence that sialylated O-glycans are involved
in mediating the contact to CLEC-2. Based on the knowledge that EDTA influences binding properties of DCSIGN [16], we next asked if also the interaction between
CLEC-2 and podoplanin depends on divalent ions. As
shown in Fig. 3D, treatment of DC-SIGN expressing cellswith EDTA significantly reduced binding to soluble
ZEBOV-GP-Fc, but had no effect on binding of soluble
podoplanin to CLEC-2 (Fig. 3D), indicating that divalentions are not required for the structural integrity of the
podoplanin binding surface of CLEC-2.
Podoplanin is incorporated into virions produced in 293T
cells and virion incorporation is essential for CLEC-2dependent HIV-1 interactions with cell lines and platelets

Our results so far indicated that podoplanin is expressedby 293T cells and that podoplanin specifically interacts
with CLEC-2. We next assessed if podoplanin is incorporated into HIV-1 released from transfected 293T cells andif the virion incorporation of podoplanin is required forHIV-1 interactions with CLEC-2. To address these questions, particularly the potential relevance of podoplaninfor HIV-1 interactions with CLEC-2, we employed
shRNA knock-down. We first tested a panel of podoplanin-specific shRNAs and identified one shRNA which

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Page 7 of 18

A)
700

150
100
50
0
10

0

10

1

10

2

10

3

10

4

% Podoplanin Expression

Counts

200

600
500
400
300
200
100
0

PDPN

293T
pcDNA3

B)

293T
PDPN

C)
450
400
350

70

p = 0.056

p = 0.039

60

Fc-Control
CLEC-2-Fc

50

300

GMCF

GMCF

p = 0.017

Isotypeanti-PDPN

250
200
150

p = 0.012

40
30
20

100

10

50

0

0

CEMx174

293T
pcDNA3

293T
PDPN

+
Fc-Control+ :DoxPDPN-Fc

Figure 2 Podoplanin is expressed on 293T cells and binds to CLEC-2. (A) 293T cells were either control transfected with empty vector or transfected with a podoplanin expression construct. Cells were stained with anti-podoplanin antibody 18H5 and analyzed by flow cytometry (black filled
area: control transfected cells stained with isotype antibody, grey filled area: control transfected cells stained with 18H5, grey line: cells transfectedwith podoplanin expression plasmid and stained with 18H5). The results of a representative experiment are shown on the left side, the average of four
independent experiments is presented at the right side. Error bars indicate SEM. (B) The experiment was carried out as described for (A), but bindingof soluble CLEC-2 to podoplanin or control transfected cells and to CEM×174 cells was analyzed. The results represent the average ± SEM of the GMCF
measured in three (CEM×174) and four (293T, 293T-PDPN) independent experiments. (C) 293 T-REx CLEC-2 cells were doxycycline treated to induce
CLEC-2 expression or PBS treated, and binding of soluble podoplanin-Fc or Fc-control protein was analyzed. The results represent the average ± SEM
of the GMCF measured in three independent experiments. Dox: doxycycline, GMCF: geometric mean channel fluorescence, PDPN: podoplanin, SEM:
standard error of the mean.

efficiently reduced podoplanin expression in transiently
transfected 293T cells (data not shown). Subsequently,
this shRNA was stably introduced into 293T cells by
employing a retroviral vector, which also contained an

expression cassette for EGFP. As control, cells were transduced with a retroviral vector encoding a non-sense
shRNA. After cultivation in selection antibiotics, all cells
were positive for EGFP and thus harboured the vector

Chaipan et al. Retrovirology 2010, 7:47
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A)

Page 8 of 18

160

PDPN-Fc Binding

CLEC-2 Expression

140

GMCF

120
100
80
60
40
20
0

ControlB)

Wt

Fc-Control
Wt

K150A E187A K190A N192A

PDPN-Fc
W

t

c1
Le

c2
Le

kDa
130
100
70

anti-PDPN

55

130
100

anti-Fc

70
55

C)

D)

60

60

p = 0.0089

50

40

40

GMCF

GMCF

50

p = 0.0733020

p = 0.147

30

PBS
EDTA

20
p = 0.028

p = 0.032

10

10

0

Wt
Fc-Control

Wt

Lec1

Lec2

0

DC-SIGN CLEC-2

PDPN-Fc

Figure 3 Binding of podoplanin to CLEC-2 requires adequate podoplanin glycosylation and is independent of divalent ions. (A) The indicated CLEC-2 mutants were transiently expressed on 293T cells and expression (white bars) and binding of podoplanin-Fc (black bars) analyzed by flow
cytometry. The results represent the average ± SEM of the GMCF measured in three independent experiments. (B) The Fc-control protein or the podoplanin-Fc fusion protein was transiently expressed in the indicated CHO cell lines. CHO Lec1 cells are defective in N-acetylglucosaminyltransferase (nocomplex N-glycans are generated), CHO Lec2 cells lack the CMP-sialic acid transporter (no sialylated glycoconjugates are generated). The supernatants of the transfected cells were harvested, concentrated and analyzed by Western blot, using the podoplanin-specific D2-40 antibody [82] (top panel) or a Fc-specific antibody (bottom panel). (C) The proteins generated in (B, control Fc-protein was 2-fold diluted) were incubated with CLEC-2
expressing 293 T-REx cells and bound protein was detected by FACS. The results represent the average ± SEM of the GMCF measured in three independent experiments. (D) Expression of DC-SIGN and CLEC-2 was induced on 293 T-REx cells by doxycycline treatment and the cells incubated withZEBOV-GP-Fc or podoplanin-Fc, respectively, in the presence of PBS (dark bars) or 2 mM EDTA containing FACS buffer (white bars). Bound proteins
were detected by flow cytometry. The results represent the average ± SEM of the GMCF measured in three independent experiments. GMCF: geometric mean channel fluorescence, PDPN: podoplanin, SEM: standard deviation of the mean.

Chaipan et al. Retrovirology 2010, 7:47
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Page 9 of 18

genome (Fig. 4A). Podoplanin expression was not appreciably altered in cells containing the vector encoding thecontrol shRNA. In contrast, cells transduced with thevector encoding the podoplanin-specific shRNA showed

substantially (~70%) reduced podoplanin expression (Fig.
4A), indicating that the shRNA was active. Next, we
tested if podoplanin was incorporated into virions
released from control cells and from the podoplanin

A)

B)
PDPN

p = 0.65150120

p = 0.006

100
50% Expression

GFP

+

-

: Virus Conc.
PDPN
p24

60
40

nonsense
shRNA

PDPN
shRNA

PDPN shRNA0

ns shRNAPDPN shRNAns shRNA

Control

+

80

20

0

-

100

Control

200

140

Mock

% Expression

250

D)
120

No shRNA100

Non-sense shRNA
10

PDPN shRNA

1

100
80
60
40
20
0

Control

CLEC-2 DC-SIGN

Transmission from
B-THP cell lines
to CEMx174 R5 cells

Direct infectionof CEMx174 R5

PDPN shRNAp = 0.0003

p = 0.016

Mock

Virus produced in 293T
expressing:1000% Transmission% Transmission

10000

Ns shRNAC)

Transmission from platelets
to CEMx174 R5 cells

Figure 4 Podoplanin is incorporated into virions released from 293T cells, and incorporation is essential for efficient CLEC-2-dependent
HIV transmission. (A) 293T cells were transduced with retroviral vectors encoding EGFP and either a podoplanin-specific or a non-sense shRNA.
Transduced cells were puromycin-selected and podoplanin (left panel) and EGFP expression (right panel) was determined by flow cytometry (using
antibody 18H5). The average ± SEM of five independent experiments, for which GMCF was determined, is presented. Podoplanin expression on cells
expressing control shRNA was set as 100%. (B) An env-defective NL4-3 proviral genome was transiently expressed in 293T cells transduced with vector
encoding either podoplanin-specific shRNA or non-sense shRNA; the supernatants were harvested, and either processed directly or concentrated bysize-exclusion filtration. Subsequently, the supernatants were analyzed for podoplanin and p24-content by Western blot. (C) The cells described in (A)were transfected with HIV-1 NL4-3 proviral DNA; the supernatants were harvested and their p24-content determined. Equal volumes of virus stocks
containing 10 ng of p24-antigen were then incubated with the indicated B-THP cell lines and bound viruses transmitted to CEM×174 R5 targets. In
parallel, direct infection of targets was assessed. The results represent the average ± SEM of six independent experiments carried out in triplicates with
two independent virus stocks. Transmission of HIV-1 produced in 293T cells not transduced with shRNA-encoding vector was set as 100%. Control
indicates B-THP cells stably transduced with empty vector. (D) The experiment was conducted as described in (C). However, HIV-1 transmission byplatelets was examined. The results represent the average ± SEM of five independent experiments carried out in triplicates. The same virus stocks asin (C) were used. Mock indicates viruses produced in 293T cells not transduced with shRNA-encoding vector. GMCF: geometric mean channel fluorescence, ns shRNA: none-sense shRNA, PDPN: podoplanin, SEM: standard error of the mean.

Chaipan et al. Retrovirology 2010, 7:47
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knock-down cells. For this, the cells were transfected with
env-deficient HIV-1 proviral DNA (for augmented biosafety), the supernatants concentrated by size-exclusion
filtration and virions pelleted by centrifugation through asucrose cushion. Alternatively, unconcentrated supernatants were directly passed through a sucrose cushion.
Western blot analysis of these virion preparations yieldeda prominent podoplanin signal for virions generated incontrol cells and a faint signal for virions generated in
podoplanin knock-down cells (Fig. 4B). These signals
were only observed for concentrated virions, and assessment of p24 content showed that concentration of particles was indeed effective (Fig. 4B). Finally, a markedly
higher podoplanin signal was measured in the supernatants of HIV transfected compared to mock transfected
cells (data not shown), confirming that the podoplaninsignal observed in Fig. 4B was mainly due to virion-associated protein. Thus, podoplanin is incorporated into
particles generated from 293T cells and incorporation
can be reduced by shRNA-mediated knock-down. We
then asked if reduced podoplanin incorporation affects
HIV-1 interactions with CLEC-2. For this, virions weregenerated in control and podoplanin knock-down cells,
normalized for p24-content and analyzed in trans-infection experiments. Reduction of virion-incorporation of
podoplanin had no effect on DC-SIGN-dependent HIV-1
transmission by B-THP cells [42] (Fig. 4C), and infection
experiments confirmed that the viruses employed were ofcomparable infectivity for target cells (Fig. 4C) and did
not infect the transmitting cells (data not shown). In contrast, diminished podoplanin incorporation resulted in apronounced reduction of viral transmission by CLEC-2
expressing B-THP cells and by platelets (Fig. 4C-D), demonstrating that podoplanin incorporation into virions
produced in 293T cells is required for efficient interaction with CLEC-2.
Reactivity of apoptotic cells with podoplanin-specificantibodies

Podocytes, which are visceral epithelial cells of the kidney, express podoplanin and were found to be infected in
HIV-1 patients and to proliferate in HIV-1 associatednephropathy [35]. We analyzed if major HIV-1 target
cells also express podoplanin. Analysis of PHA/IL-2 stimulated PBMCs and the T/B-cell hybrid cell line
CEM×174, which is permissive to HIV and SIV infection[55,56], yielded no evidence for podoplanin expression
when cells were gated for viability (Fig. 5A). Unexpectedly, however, CEM×174 cells and PBMCs defined as
non-viable by our gating strategy efficiently bound the
podoplanin antibody 18H5 but not an isotype-matched
control antibody (Fig. 5B and Additional file 1); note that
CEM×174 cells were serum starved to increase the percentage of non-viable cells. Co-staining of CEM×174 cells

Page 10 of 18

with the apoptosis marker annexin V and the necrosis
marker 7-aminoactinomycin D (7-AAD) revealed that
virtually all apoptotic cells and roughly half of thenecrotic cells reacted with the podoplanin antibody (Fig.
5B). Comparable results were obtained with PBMCs (seeAdditional file 1), albeit only a portion of the apoptotic
cells also expressed podoplanin. Apoptosis can result insurface expression of proteins which are not found on thesurface of viable cells [57,58]. It is thus possible that
podoplanin expression is up-regulated during apoptosis.
However, apoptosis can also non-specifically change antibody reactivity of cells [59]. To discern between these
possibilities, we first asked if staining of non-viable cells
was a specific feature of the particular antibody used for
detection of podoplanin (clone 18H5). Notably, stainingof apoptotic cells was also observed with a different
podoplanin antibody (clone NZ-1 [60], data not shown),
which was generated in a different species (rat) and binds
to an epitope distinct from but overlapping with the one
recognized by 18H5 [61]. In contrast, staining of apoptotic cells was not observed with several unrelated antibodies (see Additional file 2). Moreover, binding of both
antibodies, 18H5 and NZ-1, to apoptotic cells could be
inhibited by the pre-incubation of antibodies with soluble
podoplanin before staining of cells whereas pre-incubation with a control protein had no effect on antibody
binding (Fig. 5C), indicating that antibody reactivity wasdependent on the availability of the antigen binding site.
So far, we had only analyzed cells naturally undergoing
apoptosis in culture. Therefore, we next asked if reactivity
against podoplanin antibodies could be induced by triggering of apoptosis with staurosporine, a relatively nonselective protein kinase inhibitor isolated from Streptomyces staurospores [62]. Indeed, treatment of CEM×174
cells and PBMCs with staurosporine induced binding ofannexin V and anti-podoplanin-specific antibodies 18H5
and NZ-1 (Fig. 5D and Additional file 1), underlining a
potential link between apoptosis induction and podoplanin expression.
Podoplanin is not expressed on HIV-1 infected T-cells

Apoptosis of infected and bystander cells is a prominent
feature of HIV infection [63]. We therefore asked if podoplanin can be detected on HIV-1 infected C8166 T-cells
and PBMCs or on uninfected bystander cells. For this,
C8166-SEAP cells (Fig. 6A) and PBMCs (Fig. 6B) were
infected with a replication-competent HIV-1 variant harbouring EGFP and analyzed for binding of annexin V andthe podoplanin-specific antibody 18H5 at seven days post
infection, when massive cytopathic effect was visible in
infected C8166-SEAP cell cultures. Most HIV-1 infected
cells did not react with annexin V (Fig. 6, left panel), inagreement with the published observation that HIV-1
infected cells maintain phospholipid asymmetry [64].

Chaipan et al. Retrovirology 2010, 7:47
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A)

Page 11 of 18

300

700

293T

Counts225

CEMx174

150

350

75

175

0
100

102

101

103

104 100

101

102

103

PBMC

525

0
100

104

102

101

103

104

PDPN

B)

104

Annexin V

60

Dead Cells

45

1024

15

768
512
B

256
0

A

0

256

512

768

Forward Scatter

1024

102
101

0
700

100
104

55,87%

3,19%

12,26%

20,44%

Live Cells

525

103

350
175
0
100

27,99%

103

7AAD

CountsSideward Scatter

30

12,96%

10

1

10

10

2

3

10

102
101

4

100

PDPN

50,20%

100

17,11%

101

102

103

104

PDPN

C)

D)

40

3

35

Fold Increase

GMCF

30
25
20
15
10

2
1,5
1
0,5

5
0

PDPN
Annexin V

2,5

Isotype

18H5 +
18H5 + 18H5 +PBS Fc-control PDPN-Fc

0

0

2

4

6

8

Hours

Figure 5 Evidence that apoptotic cells express podoplanin. (A) Podoplanin expression on the indicated cell lines was assessed by flow cytometry.
Black filled histogram: unstained cells, grey line: cells stained with isotype control antibody, grey filled histogram: cells stained with 18H5. Similar results
were obtained in two independent experiments. (B) Apoptotic and necrotic CEM×174 express podoplanin. Cultured CEM×174 cells were serumstarved and podoplanin expression on viable and apoptotic cells, as determined by forward and sideward scatter, analyzed by flow cytometry (leftpanel, the histograms were obtained by gating on dead or live cells, as indicated by the arrows in the scatter plot). Alternatively, the cells were costained with podoplanin-specific antibody and the apoptosis marker annexin V or the necrosis marker 7-AAD, and staining analyzed by flow cytometry
including both, live and dead cells. Black filled histogram: unstained cells, grey line: cells stained with isotype control antibody, grey filled histogram:cells stained with 18H5. (C) The podoplanin-specific antibody 18H5 was pre-incubated with podoplanin-Fc fusion protein or Fc-control protein before
addition to apoptotic cells, and antibody binding was subsequently analyzed by flow cytometry. The results of a representative experiment are shown.
Similar results were obtained in three separate experiments. (D) Serum-starved CEM×174 cells were incubated with 1 μM staurosporine for the indicated times and then stained with anti-podoplanin antibody 18H5 and annexin V. Subsequently, podoplanin expression (black bars) and annexin V
binding (white bars) were determined by flow cytometry. The results represent the average of two independent experiments and are shown relative
to the podoplanin expression and annexin V binding at 0 hrs. The error bars indicate SEM. GMCF: geometric mean channel fluorescence, SEM: standard
error of the mean.

Chaipan et al. Retrovirology 2010, 7:47
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Page 12 of 18A)

C8166 T cells
104

15.7

103
102
101

8.1

103
102

2.7

21.5

54.7

10

1

10

2

10

3

10

100
100

4

3.7

68.8

10

1

103
102

10

2

10

3

10

54.6

100
100

4

GFP

GFP

B)

8.6

101

101

100
100

33.1

10

1

10

2

10

104

3

Annexin V

PBMCs
19.6

104

0.95

PDPN

103
102
101

12.8

103
102

5.2

74.3

101

102

103

104

3.41

11.5

103
102
101

101

100
100

104

1.15

PDPN

104

Annexin V

104

1.6

PDPN

27

PDPN

Annexin V

104

5.57

80.5

100
100

GFP

101

102

GFP

103

104

100

26.9

58.3

100

101

102

103

104

Annexin V

Figure 6 Podoplanin is not expressed on HIV-infected T-cells. (A) C8166-SEAP cells were infected with an HIV-1 NL4-3 variant bearing the EGFP
gene in place of nef. At 7 days post infection the infected cells were stained with podoplanin-specific antibody 18H5 and annexin V and analyzed byflow cytometry. Similar results were obtained in an independent experiment. (B) The experiment was conducted as described in (A). However, PHA
stimulated PBMCs were infected and stained. The results were confirmed in two separate experiments.

Likewise, infected cells did not bind the podoplanin-specific antibody (Fig. 6, middle panel). In contrast, podoplanin was readily detected on annexin V-positive cells (Fig.
6 right panel), which mainly represent uninfectedbystander cells (Fig. 6, left panel). These observations
suggest that podoplanin is not expressed on HIV-1
infected primary and immortalized T-cells and mightthus play a limited role in cellular attachment of HIV-1 in
infected patients.
Viruses generated in PBMCs are transmitted by CLEC-2

Our expression studies indicated that podoplanin is not
expressed on stimulated, viable PBMCs and T-cell lines(Fig. 5), and that podoplanin expression is not induced in
C8166 T-cells and PBMCs by HIV-1 infection (Fig. 5).
These results raised the question if viruses generated in
PBMCs are indeed transmitted in a CLEC-2-dependent

fashion. Notably, B-THP CLEC-2 cells promoted transinfection of HIV-1 NL4-3 (X4-tropic) produced in 293T
cells and PBMCs, and these processes could be reducedby CLEC-2-specific antiserum (Fig. 7A). Likewise, HIV-1
SF33 (X4-tropic) generated in PBMCs was transmitted toT-cells by B-THP CLEC-2 cells, and transmission was
inhibited by CLEC-2 specific antiserum to an extent
which closely approached statistical significance (Fig. 7B),
suggesting that viruses generated in PBMCs harbour acellular factor which mediates binding to CLEC-2, but isdifferent from podoplanin.

Discussion
Several cellular lectins interact with the highly glycosylated HIV Env protein [16,25,65-67], and virus capture bythese factors has been suggested to impact HIV spread inand between individuals [15,16,68]. We have previouslyChaipan et al. Retrovirology 2010, 7:47
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Page 13 of 18A)
293T-derived HIV-1

PBMC-derived HIV-1

Luciferase-Activity (c.p.s.)

1000000

100000

PBS

p = 0.005

Control Serum10000
p = 0.15

anti CLEC-2

1000

100

10

Control

CLEC-2 DC-SIGN
Transmission

B)

Control
DirectInfection

CLEC-2 DC-SIGN
Transmission

DirectInfection

PBMC-derived SF33

Luciferase-Activity (c.p.s.)

1000000

100000p = 0.053

PBS
10000

Control Serumanti CLEC-2

1000

100

10

Control

CLEC-2
Transmission

DC-SIGN
DirectInfection

Figure 7 HIV-1 produced in PBMCs is transmitted by CLEC-2. (A) Stocks of HIV-1 NL4-3 were generated in 293T cells (left panel) and in PBMCs (rightpanel) and used for transmission and direct infection, employing the indicated cell lines, as described for figure 4. The results of representative experiments performed in triplicate are shown, error bars indicate SD. Similar results were obtained in two independent experiments (B) The experiment
was carried out as described in (A) but transmission of HIV-1 SF33 generated in PBMC was analyzed. The results of a representative experiment performed in triplicate are presented and were confirmed in a separate experiment; error bars indicate SD. C.p.s.: counts per second, SD: standard deviation.

reported that platelets, anucleated cell fragments whichplay an essential role in hemostasis, express the HIV
attachment promoting proteins DC-SIGN and CLEC-2[29]. Here, we show that DC-SIGN and CLEC-2 employ
fundamentally different strategies to capture HIV. DCSIGN binds to the HIV Env protein, while CLEC-2 recognizes (a) cellular factor(s) incorporated into HIV particles. The cellular mucin-like glycoprotein podoplanin
was identified as such a factor, at least for virions generated in the widely used kidney-derived cell line 293T.
Podoplanin was not expressed on viable T-cells, themajor HIV target cell, and might thus be of minor importance for viral spread in vivo. Nevertheless, virions generated in PBMCs, which were found to be podoplanin
negative, were transmitted to T-cells in a CLEC-2-dependent fashion, suggesting that PBMC-derived particles
might harbour a so far undiscovered CLEC-2 ligand.
Finally, a potential link between podoplanin expression
and apoptosis was discovered which merits further investigation.
DC-SIGN recognizes mannose-rich carbohydrates on
the surface of the HIV Env protein and requires Ca++ ions

Chaipan et al. Retrovirology 2010, 7:47
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for its structural integrity [16-19]. Consequently, DCSIGN bound to soluble Env, binding of soluble DC-SIGN
to 293T cells was strongly enhanced by expression of HIV
Env, and ligand binding to DC-SIGN was prevented by
the mannose-polymer mannan and chelators like EDTA
(Fig. 1). In contrast, CLEC-2 did not recognize soluble
HIV Env, binding of soluble CLEC-2 to 293T cells was
not augmented by expression of HIV Env, and mannan
and EDTA did not interfere with ligand binding to CLEC2 (Fig. 1). These findings confirm our previous results
obtained with virus-particles [29] and suggest that CLEC2 does not recognize Env, but a host cell factor which isexpressed on 293T cells. They also indicate that CLEC-2
is neither mannose-specific nor calcium-dependent.
Thus, DC-SIGN and CLEC-2 differ profoundly in theirmechanisms of ligand binding and in their ligand specificities.
The discovery of Suzuki-Inoue and colleagues [53] that
podoplanin, a cellular mucin expressed on kidney podocytes [37], type I alveolar cells and lymphoid endothelial
cells [36], binds to CLEC-2 and activates CLEC-2-dependent signalling, suggested that podoplanin might be theelusive CLEC-2 ligand on 293T cells. Indeed, FACS analysis revealed robust and homogenous podoplanin expression on 293T cells (Fig. 2), in agreement with recently
published reports [69,70], and binding studies with soluble proteins confirmed that CLEC-2 and podoplanininteract (Fig. 2). Watson and colleagues previously
defined amino acids in CLEC-2, which are important for
the interaction with the snake venom component rhodocytin, and suggested that CLEC-2 binding to ligands
might be carbohydrate-independent [33,34]. Notably,
none of the amino acid residues important for rhodocytinbinding was critical for efficient binding to podoplanin,
while the presence of sialylated glycotopes on podoplanin
was indispensable (Fig. 3), in agreement with previous
results [54,71]. Rhodocytin and podoplanin might therefore engage CLEC-2 differentially, and a potential lectinactivity of CLEC-2 requires further investigation.
The endogenous expression of podoplanin on 293T
cells and the specific interaction of podoplanin with
CLEC-2 raised the questions if podoplanin was incorporated into virions produced in 293T cells, and if incorporation of podoplanin was required for CLEC-2 binding ofthese virions. Western blot analysis and knock-down of
podoplanin expression by shRNA provided affirmative
answers to both questions: Podoplanin depletion reduced
CLEC-2-, but not DC-SIGN-, dependent HIV-1 transmission by B-THP cells, and diminished transmission byplatelets by about 50% (Fig. 4). The latter finding is inagreement with our previous observation that CLEC-2specific antiserum reduced HIV-1 transmission by platelets by about half [29]. Podoplanin therefore joins the listof host factors which can be incorporated into the HIV-1

Page 14 of 18

envelope and impact HIV-1 infection by interacting with
their cognate ligands [9,10]. A prominent example forsuch a factor is ICAM-1 which was found to be incorporated into the viral membrane, and to facilitate HIV-1
infection by binding to its ligand LFA-1 on T-cells [12].
The potential relevance of podoplanin incorporationfor HIV spread in infected individuals is critically determined by the overlap of the podoplanin expression pattern with the cellular tropism of HIV. Analysis of T-celllines and PBMCs for podoplanin expression yielded negative results (Fig. 5), at least when viable cells were analyzed (see below), indicating that HIV particles generated
in patients might not harbour podoplanin. The exception
might be viruses released from kidney podocytes whichhave been documented to express podoplanin [37] and tobe susceptible to HIV infection [35]. However, the biological relevance of this process is questionable. In this context, it also needs to be noted that podoplanin expression
is up-regulated in many tumours including Kaposi sarcoma [72,73]. Podoplanin/CLEC-2-dependent platelet
stimulation by tumour cells promotes hematogenous
tumour metastasis [71,74], possibly by inducing growth
factor secretion by platelets and by promoting formationof a "platelet cap", which protects the tumour frommechanical forces. Thus, podoplanin might play a role inthe development of the AIDS-associated Kaposi sarcoma,
but is unlikely to modulate HIV spread in patients. Nevertheless, HIV-1 produced in PBMCs was transmitted totarget cells in a CLEC-2-dependent fashion (Fig. 7), suggesting that primary T-cells might express a so far unrecognized CLEC-2 ligand (a hypothesis also raised byothers [75]), which is incorporated into the viral envelope
and which facilitates HIV transmission by CLEC-2. Our
ongoing studies are devoted to the identification of thisfactor.
Podoplanin was not detected on viable CEM×174 cells(a T/B cell hybrid) and PBMCs, as determined by our gating strategy and by co-staining with the apoptosis andnecrosis markers annexin V and 7-AAD, respectively(Fig. 5 and Additional file 1). In contrast, we observed
efficient reactivity of two different podoplanin antibodies
with non-viable cells, raising the intriguing possibility
that podoplanin might be expressed at the cell surface inthe context of apoptosis. Apoptosis can indeed alter
expression of surface markers [57,58,76] but might also
modulate antibody reactivity of cells [59], making theanalyses of podoplanin expression by apoptotic cells atechnically challenging task. Our findings that two antibodies, 18H5 and NZ-1, which were generated in different species and recognize different but overlapping
epitopes in podoplanin [61], both specifically bind to
apoptotic cells (Fig. 5 and data not shown), and that thisreactivity depends on the availability of the antigen-binding site (Fig. 5C) suggests to us that binding is most likely

Chaipan et al. Retrovirology 2010, 7:47
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specific. Furthermore, nested RT-PCR detected podoplanin message in CEM×174 cells (data not shown), suggesting low levels of podoplanin expression in these cells.
Importantly, the podoplanin message did not appreciably
increase upon apoptosis induction, and treatment withcycloheximide did not block specific staining of apoptotic
cells with podoplanin antibodies (data not shown).
Therefore, one must assume that podoplanin protein (oran antigenically related protein) is present within
CEM×174 cells and other cell types, and that the protein
becomes accessible to antibody staining only upon induction of apoptosis. If the latter process is due to specifictransport of podoplanin to the cell surface or to membrane disintegration during apoptosis could not be conclusively determined. Regardless of the mechanism
underlying reactivity of apoptotic cells with podoplaninspecific antibodies, podoplanin was not detected on HIV
infected viable and apoptotic cells (Fig. 6), indicating that
podoplanin expression is not altered in the context of
HIV infection.
Collectively, our data help to understand how HIVinteracts with CLEC-2, an HIV attachment factor onplatelets. Several lines of evidence suggest that this interaction could impact HIV spread in infected patients. For
one, thrombocytopenia (reduced platelet count) is frequent in HIV/AIDS patients [77], and it is conceivablethat CLEC-2-dependent binding of HIV to platelets
results in platelet clearance and thus contributes toreduced platelet counts. In addition, the interaction of
HIV with CLEC-2 on platelets might induce platelet activation, which was found to be associated with HIV infection [78]. Moreover, CLEC-2-dependent HIV binding toplatelets might result in trans-infection or virus degradation [28,29], and both processes could impact viral loadand disease development. Finally, it is worth noting thatliver sinusoidal endothelial cells and megakaryocytes also
express CLEC-2 [66] and that both cell types are susceptible to HIV infection [79-81], which might be modulatedby CLEC-2. In summary, CLEC-2 is expressed on several
cell types exposed to HIV in patients and thus has thepotential to modulate viral spread.

Conclusions
Our results highlight that incorporation of cellular factors
can alter HIV attachment to cells and cell to cell transmission. While podoplanin is unlikely to be incorporated
into HIV particles produced in infected patients, ourresults indicate that HIV might incorporate a functional
analogue of podoplanin in vivo, and that this process
might promote virus binding to CLEC-2 positive cells.
The identification of the respective factor and the clarification of the potential connection between podoplanin

Page 15 of 18

expression and apoptosis are interesting tasks for future
research.

Additional material
Additional file 1 Evidence that apoptotic PBMCs express podoplanin.
(A) Apoptotic and necrotic PBMCs express podoplanin. Podoplanin expression on viable and apoptotic PBMCs, as determined by forward and sideward scatter, was analyzed by flow cytometry (left panel, the histograms
were obtained by gating on dead or live cells, as indicated by the arrows in
the scatter plot). Alternatively, the cells were co-stained with podoplaninspecific antibody and the apoptosis marker annexin V or the necrosis
marker 7-AAD, and staining analyzed by flow cytometry including both, live
and dead cells. Black filled histogram: unstained cells, grey line: cells stainedwith isotype control antibody, grey filled histogram: cells stained with 18H5.
(B) PBMCs were incubated with 1 μM staurosporine for the indicated times
and podoplanin expression (black bars) and annexin V binding (white bars)
were determined by flow cytometry. The results were confirmed in two
independent experiments.
Additional file 2 The podoplanin-specific antibody 18H5, but not
antibodies with other specificities recognize non-viable cells. (A)CEM×174 cells were analyzed for their distribution in the forward and sideward scatter, and a gate was defined which comprised both viable andnon-viable cells. (B) The CEM×174 cells were stained with the indicatedmonoclonal antibodies and staining of the cells gated as shown in (A) was
analyzed. The results of a representative experiment are shown and wereconfirmed in an independent experiment. IgG1, IgG2a and IgG2b are commercially available isotype control antibodies. The anti-AU1 antibody is specific for the AU1 antigenic tag. ACE2, MER and Axl are cell surface receptors,
which are used for cell entry by SARS-coronavirus (ACE2) and Ebola virus(Axl, MER). PDPN: podoplanin.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CC analyzed binding of soluble CLEC-2 and soluble DC-SIGN to HIV Env, determined podoplanin expression by 293T cells, generated and characterized
podoplanin knock-down cells, compared transmission of HIV generated in
podoplanin-knock-down and control cells, and analyzed CLEC-2-dependenttransmission of HIV generated in 293T cells and PBMCs, IS analyzed podoplanin
expression on apoptotic cells, determined inhibition of antibody binding to
apoptotic cells by soluble podoplanin, analyzed podoplanin expression on
uninfected cell lines and HIV infected C8166-SEAP cells, TST analyzed bindingof soluble podoplanin to CLEC-2 mutants, determined the impact of glycosylation and divalent ions on podoplanin binding to CLEC-2, analyzed podoplanin
expression on cell lines and determined podoplanin incorporation into virions,
SB and IG determined podoplanin RNA expression in apoptotic cells and analyzed podoplanin expression by viable cells, JS analyzed podoplanin expression on HIV infected PBMCs, YK provided critical reagents and contributed tothe interpretation of experiments, JM, GS and RGS contributed to the design
and the interpretation of experiments, SP planned and supervised the research
and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank B. Fleckenstein, K. von der Mark and T.F. Schulz for support. This work was supported by the DFG (grants GK 1071 and SFB466 to SP),BMBF (to SP), the MD/PhD program Molecular Medicine (to TST), and the Center for Infection Biology (to I.S.)
Author Details
1Nikolaus-Fiebiger-Center for Molecular Medicine, University Hospital Erlangen,91054 Erlangen, Germany, 2Institute for Clinical and Molecular Virology,
University Hospital Erlangen, 91054 Erlangen, Germany, 3Institute of Virology,
Hannover Medical School, 30625 Hannover, Germany, 4The Global COE
Program for Medical Sciences, Japan Society for the Promotion of Science, TheChaipan et al. Retrovirology 2010, 7:47
http://www.retrovirology.com/content/7/1/47

Oncology Research Center, Research Institute for Advanced Molecular
Epidemiology, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan, 5Institute of Molecular Virology, University Hospital Ulm, 89081 Ulm, Germany, 6Blood Systems Research Institute and Department of Laboratory Medicine,
University of California, San Francisco, CA, USA and 7Cellular Chemistry, Center
for Biochemistry, Hannover Medical School, 30625 Hannover, Germany
Received: 11 December 2009 Accepted: 19 May 2010
Published: 19 May 2010©
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Cite this article as: Chaipan et al., Incorporation of podoplanin into HIV
released from HEK-293T cells, but not PBMC, is required for efficient binding
to the attachment factor CLEC-2 Retrovirology 2010, 7:47

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