Two Key Residues in EphrinB3 Are Critical for Its Use as an Alternative Receptor for Nipah Virus

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DOI: 10.1371/journal.ppat.0020007 · Source: PubMed
Abstract
EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.
Two Key Residues in EphrinB3 Are Critical
for Its Use as an Alternative Receptor
for Nipah Virus
Oscar A. Negrete
1
, Mike C. Wolf
1
, Hector C. Aguilar
1
, Sven Enterlein
2
, Wei Wang
3
, Elke Mu
¨
hlberger
4
, Stephen V. Su
1
,
Andrea Bertolotti-Ciarlet
5
, Ramon Flick
2
, Benhur Lee
1,6,7*
1 Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles , California, United
States of America, 2 University of Texas Medical Branch, Galveston, Texas, United States of America, 3 Department of Medicine-Infectious Diseases, David Geffen School of
Medicine, University of Cal ifornia Los Angeles, Los Angeles, California, United States of America, 4 Institute for Virology, Philipps-University Marburg, Marburg, Germany,
5 Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 6 Department of Pathology and Laboratory Medicine, David
Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 7 UCLA AIDS Institute, David Geffen School of Medicine,
University of California Los Angeles, Los Angeles, California, United States of America
EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus.
Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping
expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we
show that of all known ephrins (ephrinA1–A5 and ephrinB1–B3), only the soluble Fc-fusion proteins of ephrinB3, in
addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3
and B2 with subnanomolar affinities (K
d
¼ 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon
resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate
(K
off
¼ 1.94 3 10
3
s
1
versus 1.06 3 10
4
s
1
for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral
entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral
entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an
overlapping site. Mutational analysis indicated that the Leu–Trp residues in the solvent exposed G–H loop of ephrinB2
and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr–Met residues in the
homologous positions in ephrinB1 with Leu–Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona
fide alternate receptor for NiV entry, and two residues in the G–H loop of the ephrin B-class ligands are critical
determinants of NiV receptor activity.
Citation: Negrete OA, Wolf MC, Aguilar HC, Enterlein S, Wang W, et al. (2006) Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus.
PLoS Pathog 2(2): e7.
Introduction
Nipah virus (NiV) is a zoonotic paramyxovirus classified in
the taxonomic unit Henipavirus under the family of Para-
myxoviridae [1,2]. NiV emerged in peninsular Malaysia and
Singapore in 1998–1999 when cases of severe acute encepha-
litis occurred among agricultural and abattoir workers in
close contact with NiV-infected pigs [3]. In 2004 two
confirmed outbreaks of NiV in Bangladesh recorded mortal-
ity rates of greater than 70% with evidence of human-to-
human transmission [4,5]. Due to high mortality rates and its
potential use as a bioweapon [6], NiV has been classified as a
Category C priority pathogen for biodefense purposes. NiV
not only has the potential to cause severe disease in humans,
but also represents a critical economic threat if used against
the pig-farming industry.
Pathological investigations of NiV-infected patients re-
vealed that a major cellular target of the NiV appears to be
endothelial cells that line blood vessels [7]. More important,
syncytial or multinucleated giant endothelial cells were seen
in the microvasculature of many organs, with the most severe
damage occurring to vessels in the central nervous system
(CNS). Concordantly, NiV antigen load was highest in the
brain parenchyma, especially in neurons, compared with
other organs. The functional receptor for NiV entry, ephrinB2
[8,9], is expressed on endothelial cells and neurons [10,11]
consistent with the known cellular tropism for NiV [7,12].
Ephrins are the highly conserved ligands to the Eph family
of receptor tyrosine kinases [13]. Eph–ephrin signaling
functions in both embryonic and adult tissues by regulating
processes such as angiogenesis, neuron axonal guidance, and
tumorgenesis [11,13–15]. Both ephrins and Eph receptors are
categorized into class-A and class-B protein s bas ed on
sequence homology, binding affinities, and the manner of
ephrin membrane attachment. In general, ephrin ligands and
receptors can interact promiscuously inside their own class
Editor: Grant McFadden, Robarts Research Institute, Canada
Received December 13, 2005; Accepted December 30, 2005; Published February
10, 2006
DOI: 10.1371/journal.ppat.0020007
Copyright: Ó 2006 Negrete 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.
Abbreviations: CHO, Chinese hamster ovary; CNS, central nervous system; ELISA,
enzyme-linked immunosorbent assay; Luc, luciferase; MOI, multiplicity of infection;
NiV, Nipah virus; NiV-G, NiV attachment protein G; RFP, red fluorescent protein;
VSV, vesicular stomatitis virus
* To whom correspondence should be addressed. E-mail: bleebhl@ucla.edu
PLoS Pathogens | www.plospathogens.org February 2006 | Volume 2 | Issue 2 | e70078
but not outside of it. An exception to the rule is the binding
of EphA4 to ephrinB3, an interaction thought to be
important in corticospinal neurons crossing the spinal cord
midline [16,17]. Ephrins have extremely conserved features,
especially within a class, and can have overlapping but
distinct expression patterns [18].
Here, we examined whether the NiV attachment protein G
(NiV-G) can act as promiscuously as the Eph receptors in
binding to other ephrins, in addition to ephrinB2. We showed
that ephrinB2 and B3 bound to NiV-G with subnanomolar
affinities. Expression of human ephrinB3 in nonpermissive
CHO-pgsA745 cells rendered them permissive to NiV entry
and infection. Lastly, we characterized the important
conserved features of ephrinB2 and B3 that account for their
NiV receptor activity. The discovery of a more comprehen-
sive set of NiV receptors will aid our understanding of the
pathology underlying NiV disease.
Results
NiV-G Binds EphrinB3 at Lower Affinity than EphrinB 2
Ephrins constitute a highly conserved class of proteins with
many homologous me mbers. Thus, we examined if any
ephrins, other than ephrinB2, can bind similarly to NiV-G.
Using an enzyme-linked immunosorbent assay (ELISA), we
screened for the ability of soluble HA-tagged ectodomain of
NiV-G (NiV-G-HA) to bind to all known ephrins (ephrinA1–
A5 and ephrinB1–B3). We found that ephrinB3-Fc, in
addition to ephrinB2-Fc, bound to NiV-G-HA (Figure 1A).
To confirm these results with cell surface-expressed ephrins,
CHO-pgsA745 cells were stably transfected wit h human
ephrinB1 (CHO-B1), ephrinB2 (CHO-B2), o r ephrinB3
(CHO-B3). Chinese hamster ovary (CHO) cells do not express
ephrins endogenously [19], while the CHO-pgsA745 cells
derived from these CHO cells lack heparin sulfate proteo-
glycans [20]. Heparin sulfates are known to act as entry or
attachment receptors for many viruses, which can confound
viral receptor studies. Thus, we used NiV-G-Fc, a fusion
construct between the ectodomain of NiV-G and the Fc
region of human IgG1, to measure the binding of NiV-G to
each of the ephrin B-class ligands stably expressed on CHO-
pgsA745 cells (Figure 1B). Remarkably, the K
d
for NiV-G-Fc
binding to ephrinB2 and B3 was in the subnanomolar range
(K
d
¼ 0.06 nM and 0.58 nM for ephrinB2 and B3, respectively).
NiV-G-Fc did not bind to CHO-B1 cells when tested under
the same conditions used for CHO-B2 and CHO-B3 cells
(unpublished data).
To delineate the difference in binding affinities between
ephrinB2 and B3, we examined their binding kinetics to NiV-
G-Fc. We performed surface plasmon resonance analysis by
coupling NiV-G-Fc to the sensor chip (as ligand) while using
soluble ephrinB2 and B3 as analyte. BIAcore analysis of NiV-
G-Fc binding to ephrinB2-Fc and B3-Fc indicated that while
NiV-G-Fc bound to ephrinB2 and B3 with similar on-rates
(K
on
¼ 9.69 3 10
5
and 6.87 3 10
5
for ephrinB2 and B3,
respectively), their off-rates were significantly different
(Figure 1C and 1D). The K
off
for ephrinB2-Fc binding to
NiV-G-Fc (1.06 3 10
4
s
1
) was more than 10-fold slower than
ephrinB3-Fc (1.94 3 10
3
s
1
) binding (Figure 1D). EphrinB1-
Fc was also tested similarly to ephrinB2 and B3 and did not
exhibit any binding to NiV-G-Fc even up to concentrations as
high as 500 nM (unpublished data). As a control to determine
the accuracy of our BIAcore measurements, we determined
the K
d
of EphB4-Fc binding to ephrinB2-Fc to be 0.37 nM, a
value consistent with published values of approximately 0.5
nM [21] (unpublished data). Cumulatively, our data show that
the Nipah attachment protein bound to both ephrinB2 and
ephrinB3 with different but significant affinities.
EphrinB3 Supports NiV Entry and Infection
We next examined whether the high-affinity protein
interaction seen between ephrinB3 and NiV-G was sufficient
to permit the entry of NiV. We first established a panel of
CHO-pgsA745 cells stably expressing ephrinB1, B2, and B3.
Since the EphB3 receptor binds to all ephrin B-class ligands
with similar affinities (0.27–1.8 nM for ephrinB1, 0.28–0.78
nM for ephrinB2, and 1.5 nM for ephrinB3) [21], we used
saturating amounts of soluble EphB3 (EphB3-Fc) to deter-
mine the level of ephrinB1–B3 expression in our stable cell
lines by flow cytometry (Figure 2A). We found that while
CHO-B1, CHO-B2, and CHO-B 3 cells were significantly
positive for EphB3 binding (68%, 47%, and 47%, respec-
tively), only CHO-B2 and CHO-B3 cells bound to NiV-G-Fc
(46% and 31%, respectively). Soluble EphB3 did not bind to
parental CHO-pgsA745 cells, nor did the ephrin A-class-
specific EphA2-Fc bind any of the cell lines tested (CHO-
pgsA745, CHO-B1, CHO-B2, or CHO-B3). These resul ts
confirm the specific ephrin B-class expression on our panel
of CHO-pgsA745 cells.
We then proceeded to quantitate NiV entry using NiV
envelope pseudotyped luciferase (Luc) reporter viruses. We
had previously shown that NiV envelopes can be successfully
pseudotyped onto recombinant vesicular stomatitis virus
(VSV) expressing a red fluorescent protein (RFP), but lacking
its own envelope (NiV-VSV-DG-RFP) [8]. Here, the NiV-VSV-
DG-Luc was made bearing the NiV fusion (F) and NiV-G and
expressing the Renilla Luc reporter gene in place of the RFP
gene. These NiV envelope pseudotyped VSV particles were
used to infect CHO-pgsA745 parental cells (CHO), CHO-B1,
CHO-B2, and CHO-B3 cell lines. We found that both CHO-
B2 and CHO-B3 allowed entry of NiV-VSV-DG-Luc virus
PLoS Pathogens | www.plospathogens.org February 2006 | Volume 2 | Issue 2 | e70079
Alternative Receptor for Nipah Virus
Synopsis
Nipah virus is a deadly virus that can cause death in up to 70% of
infected patients, mostly from fatal inflammation of the brain. Nipah
virus is considered a ‘‘ priority pathogen’’ for bioterrorism purposes,
and it has the potential for widespread economic devastation as it
can spread rapidly among susceptible livestock. The authors had
previously identified the receptor that mediates Nipah virus entry
into cells. This receptor, ephrinB2, is a critical molecule for the
development of the vascular and nervous system and is highly
expressed on endothelial cells and neurons, which are also the two
cell types preferentially infected by Nipah virus in vivo.
EphrinB2 belongs to a large family of related molecules that are
variably conserved in structure and function. Thus, the authors
screened all known ephrins, and found that a closely related
molecule, ephrinB3, also can function as an entry receptor for Nipah
virus. In addition, the authors established that while ephrinB2 was
better used than ephrinB3 as an entry receptor, the same two critical
amino acids in ephrinB2 and B3 were responsible for the viral
receptor activity of these molecules. The discovery of a more
comprehensive set of NiV receptors will aid our understanding of
the pathology underlying NiV disease.
(Figure 2B), although viral entry into CHO-B2 cells reprodu-
cibly resulted in higher Luc levels (p ¼ 0.05, paired t-test). In
three independent experiments, the reduction in viral entry
into CHO-B3 cells ranged from 21% to 46%. Since the
EphB3-Fc binding data indicated similar levels of ephrinB2
and B3 expression on the CHO-B2 and CHO-B3 cells (Figure
2A), NiV-VSV-DG-Luc virus entered CHO-B2 cells more
efficiently than CHO-B3 cells.
To examine whether ephrinB3 can support viral infection,
live NiV infections were performed under Biosafety Level-4
conditions. The hallmark of NiV infection in humans is the
presence of syncytial or multinucleated giant endothelial
cells, and cell lines from many different species produced
syncytia upon infection. Therefore, we looked for syncytia
formation in CHO, CHO-B1, CHO-B2, CHO-B3, and Vero
cells after infection with live NiV. Indeed, we found that live
NiV can infect ephrinB2- and B3-expressing cells, although
ephrinB2 appears to be used more efficiently (Figure 2C).
With any given multiplicity of infection (MOI), at 24 h
postinfection, there were always a greater number of syncytia
in the CHO-B2 versus CHO-B3 cells. No syncytia were
detected in CHO-B1 cells. At 48 h postinfection, syncytia
were apparent at all MOIs tested in the CHO-B3 cells
(unpublished data). Thus, ephrinB3 can serve as a bona fide
alternative receptor for NiV entry.
NiV-G Binds EphrinB2 and B3 via an Overlapping Site
Next, we asked whether ephrinB2 and B3 interact with
NiV-G in a distinct or overlapping manner. To answer this
question, we used a competition assay where CHO-B2 and
CHO-B3 cells were infected with NiV-VSV-DG-Luc viruses in
the presence of soluble ephrin B-class ligands. As expected,
ephrinB2-Fc inhibited pseudotyped NiV on CHO-B2 cells
while ephrinB1-Fc did not inhibit entry (Figure 3, left).
However, ephrinB3-Fc also inhibited pseudotyped NiV entry
on CHO-B2 cells, suggesting that ephrinB3 blocked eph-
rinB2-dependent NiV entry by competing for a similar
binding domain on NiV-G. Conversely, ephrinB2-Fc also
Figure 1. Soluble NiV-G Binds to EphrinB3 with Lower Affinity than EphrinB2
(A) 1.0 lg/ml, 0.1 lg/ml, and 0.01 lg/ml of the indicated ephrin-Fc fusion proteins were allowed to bind to soluble NiV-G-coated plates in an ELISA
format (see Materials and Methods). The amount of ligand bound was detected colorimetrically using an antihuman Fc antibody conjugated to
horseradish peroxidase. One representative experiment out of three is shown. Data are averages of triplicates 6 standard error (SE).
(B) EphrinB2 and B3 stably transfected CHO-pgsA745 cells (CHO-B2 and CHO-B3, respectively) were used to measure NiV-G-Fc cell surface binding.
Increasing concentrations of NiV-G-Fc were added to either CHO-B2 cells (dashed line with squares) or CHO-B3 cells (solid line with triangles), and
binding was assessed by flow cytometry using R-phycoerythrin-conjugated anti-Fc antibodies. Regression curves were generated as described in
Materials and Methods. Each data point is an average 6 SE from three experiments.
(C) Surface plasmon resonance (BIAcore 3000) measured the binding kinetics of NiV-G-Fc to both ephrinB2-Fc and ephrinB3-Fc in response units (RU).
NiV-G-Fc was immobilized to a CM5 sensor chip via an amide coupling procedure, and increasing concentrations of ephrinB2-Fc and ephrinB3-Fc were
flowed as analyte over the sensor chip. One representative experiment out of two is shown.
(D) K
d
,K
on
(association-rate), and K
off
(dissociation-rate) were determined by fitting the binding chromatogram data from (C) with BIAcore evaluation
software (version 3.1) using the 1:1 Langmuir binding model.
DOI: 10.1371/journal.ppat.0020007.g001
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Alternative Receptor for Nipah Virus
inhibited pseudotyped NiV entry on CHO-B3 cells (Figure 3,
right). In both CHO-B2 and CHO-B3 cells, ephrinB2-Fc was a
more effective inhibitor of NiV-G entry than ephrinB3-Fc. In
summary, these results suggest that ephrinB2 and B3 binding
sites on NiV-G are overlapping.
Leu–Trp Residues in the G–H Loop of EphrinB2 and B3 Are
Critical for NiV-G Binding
Since ephrinB2 and B3 can support NiV entry, while
ephrinB1 cannot, we hypothesize that conserved residues
common in both ephrinB2 and B3 mediate specific inter-
actions with NiV-G. Alignment of human, mouse, and rat
ephrinB1, B2, and B3 sequences identified common residues
in ephrinB2 and B3 not present in ephrinB1 (Figure 4A).
Examination of the homologous G–H loop regions between
ephrinB1, B2, and B3 reveals that the L–W (Leu–Trp) residues
present in ephrinB2 and B3 are replaced by Y–M (Tyr–Met) in
ephrinB1 (Figure 4A). The cocrystal structure of ephrinB2
and the EphB2 receptor (an endogenous ephrinB2 receptor)
indicates that the L–W (Leu
124
–Trp
125
) residues in the G–H
loop of ephrinB2 insert deep into a hydrophobic pocket in
EphB2 [22]. We believe this interaction to be informative as
soluble EphB2 inhibits NiV-G mediated infection[8] and is
likely to interact with a similar region on ephrinB2 as NiV-G.
Thus, we sought to determine if the G–H loop L–W residues
in ephrinB2 and ephrinB3 also interact with NiV-G.
Full-length human ephrinB1, B2, and B3 clone s were
obtained, and the ectodomain of each was fused to the Fc
region of human IgG1. Replacement of Y–M in ephrinB1 with
L–W from the homologous positions in ephrinB2 and B3
resulted in a soluble ephrinB1 mutant, B1
LW
-Fc, that bound
NiV-G almost as well as ephrinB2-Fc (Figure 4B). Conversely,
the L–W to Y–M mutations in soluble ephrinB2 (B2
YM
-Fc) and
ephrinB3 (B3
YM
-Fc) abrogated binding to NiV-G-HA,
although at higher concentrations, B2
YM
-Fc appears to retain
minimal NiV-G binding activity.
Leu–Trp Residues in the G–H Loop of EphrinB3 Are
Necessary for NiV Entry
Next, we examined the effects of these L–W/Y–M mutations
in the context of full-length ephrins and their ability to
support NiV entry. To do so, we first made stable CHO-
pgsA745 cells expressing the full-length ephrin mutants
(CHO-B1
LW
, CHO-B2
YM
, and CHO-B3
YM
). We compared
the level of mutant ephrin expression in the stable cell lines
to that of wild-type ephrin expression using EphB3-Fc in a
Figure 2. Pseudotyped and Live NiV Use EphrinB2 and B3 for Cellular Entry
(A) Ephrin expression was measured by flow cytometry on CHO-pgsA745 parental cells (CHO) and CHO-pgsA745 cells stably expressing ephrinB1, B2,
and B3 (CHO-B1, CHO-B2, and CHO-B3). To bind the CHO cell lines, 10 lg/ml of EphA2, 10 lg/ml EphB3-Fc, and 1 nM of NiV-G-Fc were used, and the
amount of binding was detected by flow cytometry as in Figure 1B. Data are representative of three experiments.
(B) NiV-F and G glycoproteins were pseudotyped onto a VSV-DG-Luc core virus (NiV-VSV-DG-Luc) and used to infect parental CHO-pgsA745 (CHO), CHO-
B1, CHO-B2, and CHO-B3 cells. Entry of the indicated dilutions of NiV-VSV-DG-Luc viruses was measured by quantifying Renilla Luc activity according to
manufacturer’s directions. Relative light units (RLU) were acquired and quantified on a Veritas luminometer. Data are shown as averages of triplicates 6
standard deviation of a representative experiment. In three independent experiments, viral entry into CHO-B3 cells was reduced by 21%, 28%, and 46%,
respectively, compared to CHO-B2 cells (p ¼ 0.05, paired t-test).
(C) The listed MOIs of live NiV were used to infect the indicated cell lines (10
5
cells per infection). Foci of syncytia were observed 24 h postinfection. Vero E6
cells are fully permissive for NiV infection and were used as positive control cells. Note the larger number of syncytia seen on CHO-B2 versus CHO-B3 cells.
DOI: 10.1371/journal.ppat.0020007.g002
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Alternative Receptor for Nipah Virus
flow cytometry analysis. It is unlikely that EphB3 binds to the
ephrin LW/YM residues in question since EphB3 can bind
ephrinB1, B2, and B3 with similar affinities [21]. Therefore,
using EphB3-Fc to measure cell surface expression, we found
that both wild-type ephrin and its relevant mutant were
expressed at similar levels (Figure 5A; compare B1 to B1
LW
,
B2 to B2
YM
, and B3 to B3
YM
). Parental CHO-pgsA745 (CHO)
served as a negative control and soluble EphB3 did not bind
to these cells. In addition, NiV-G-Fc bound to the mutant
ephrins in the expected patterns seen in our previous solid
state ELISA experiment (Figure 4B).
We then proceeded to infect these cells with NiV-VSV-Luc
pseudotyped viruses (Figure 5B). As expected, both ephrinB2
and B3 permitted NiV envelope-mediated entry as described
previously (Figure 2B). The B1
LW
mutant now supported NiV
entry, while entry into B2
YM
cells was markedly reduced.
Entry into B3
YM
was completely abrogated. We note that
while entry into B2
YM
cells was reduced by about 85%
compared to wild-type ephrinB2 cells, entry was still 44-fold
over background (parental CHO-pgsA745 cells). These results
suggest that while the L
124
–W
125
residues in ephrinB3 appear
to be critical for NiV entry, other residues in ephrinB2 likely
have a supporting role in mediating NiV entry. Interestingly,
the B1
LW
mutant could not fully restore entry equivalent to
wild-type B2 or B3 levels, even though NiV-G bound to cell
surface ephrinB1
LW
at wild-type ephrinB2 levels. Therefore,
in addition to simple binding, additional residues in
ephrinB2 likely mediate the subsequent conformational
changes in NiV-G and/or F that leads to membrane fusion
and entry.
Discussion
EphrinB2 was recently identified as a functional cellular
receptor for NiV [8,9]. EphrinB2 expression on endothelial
cells, neurons and smooth muscle cells [10,11] is highly
consistent with the known tropism of NiV infection [7]. Here,
we show that ephrinB3 is an alternate receptor for NiV and is
independently able to support NiV entry and infection, albeit
less efficiently than ephrinB2. NiV-G binds to both ephrinB2
and B3 with subnanomolar affinity, with the relatively weaker
K
d
of NiV-G for ephrinB3 explained by its faster off-rate.
Finally, we implicate two residues (L–W) common in the G–H
loop of ephrinB2 and B3 as crucial for NiV receptor activity.
Remarkably, replacement of the Y–M residues in the
homologous positions in ep hrinB1 wi th L–W conferred
wild-type NiV-G binding activity and substantial NiV recep-
tor activity to a protein that is otherwise nonfunctional as a
NiV receptor.
To our knowledge, there is no specific indication that
ephrinB3 is expressed in the endothelium. At the minimum,
ephrinB3 does not appear to be critical to vascular develop-
ment since ephrinB3 knockout mice lack the overt defects in
vascular morphogenesis seen in ephrinB2 knockout mice
[23,24]. However, NiV entry into microvascular endothelial
cells is almost completely abrogated by soluble ephB4-Fc [8],
which binds to ephrinB2 but not B3, suggesting that ephrinB3
is likely not expressed on endothelial cells, at least not at
levels that can support robust viral entry. In contrast,
ephrinB3 is expressed in the CNS in overlapping and distinct
patterns with ephrinB2 [18,25]. In the regions of the adult
brain such as the cerebral cortex [26,27] and the hippo-
campus [28] where ephrinB2 and B3 exhibit overlapping
expression, NiV could potentially use either receptor for
entry with a possible preference for ephrinB2 based on the
higher affinity of NiV-G for ephrinB2. However, in regions
such as the corpus callosum [27] and the spinal cord [16,17],
ephrinB3 is distinctly express ed and could account for
specific aspects of NiV pathology.
EphrinB3 knockout mice studies indicate ephrinB3 is
expressed in the spinal cord midline and functions to prevent
corticospinal tract axons from recrossing the midline.
Coincidently, in a histological study of NiV infection by
Wong et al., three of eight patients examined showed
pathological lesions in the spinal cord similar to other
regions of the CNS [7]. Indeed, clinical symptoms of
segmental myoclonus and flaccid tetraplegia combined with
nerve conduction studies have also suggested upper cervical
and lower spinal cord involvement [29], and magnetic
Figure 3. EphrinB2 and B3 Bind NiV-G at an Overlapping Site
NiV-VSV-DG-Luc pseudotyped viruses were used to infect CHO-B2 and CHO-B3 cells in the presence of the indicated amounts of ephrinB1, B2, and B3-Fc
fusion proteins (B1-Fc, B2-Fc, and B3-Fc, respectively). Entry was measured as in Figure 2A. Data are the average of triplicates 6 standard deviation, and
one representative experiment of three is shown.
DOI: 10.1371/journal.ppat.0020007.g003
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Alternative Receptor for Nipah Virus
resonance imaging confirmation of a spinal cord lesion at the
cognate C7 level in a patient who developed left-arm
dysaesthesia and finger weakness has also been reported
[30]. In another study that reported magnetic resonance
imaging findings in eight patients with NiV encephalitis, half
the patients had numerous punctate lesions in the corpus
callosum [31], where ephrinB3, but not ephrinB2, is expressed
[27]. Thus, although ephrinB2 seems to be the primary
receptor for NiV, ephrinB3 can likel y be used as an
alternative receptor and may account for some of the CNS
pathology seen in NiV infection.
In this study, we also established that NiV-G binding to
ephrinB2 and B3 is dependent on the same L–W residues that
are important for endogenous ephrinB2/EphB2 interactions
[21,22]. Since NiV-G interacts with ephrinB2 in a similar
fashion with at least some of the Eph B-class receptors, and
NiV-G forms higher order oligomers [32] analogous to Eph B-
class receptors [10,11], NiV-G could potentially induce
‘‘ reverse-signaling’’ upon ephrinB2 or B3 binding.
In vivo, Eph–ephrin interactions cause bidirectional signal-
ing that can direct the migration of endothelial cells and
neuronal dendrites [10,11,33–35]. Therefore, NiV infection
may not only target ephrinB2- or B3-expressing cells, but also
disru pt normal Eph–ephrin signaling and possi bly alter
cellular migration patterns. Indeed, infusion of soluble
EphB2-Fc has been reported to disrupt the migration of
Figure 4. The Leu–Trp Residues Present in the G–H Loop of EphrinB2 and B3 Are the Critical Determinants of NiV-G Binding
(A) Sequence alignment of human (hu), mouse (ms), and rat (rt) ephrinB1, B2, and B3 ectodomains using the Jotun Hein algorithm (DNAstar Megalign
software). Six residues in the ephrin B-class ectodomain reveal solvent-exposed amino acids [22] that contain identical residues in both ephrinB2 and B3
but different residues in ephrinB1 (open box). Examination of the ephrin binding loop (G–H loop) indicates the L–W residues in ephrin B2 and B3 are
replaced by Y–M residues in ephrinB1 (filled box).
(B) Ephrin-Fc mutants were created by substituting the L–W residues present in ephrinB2 and B3 with Y–M residues using site-directed mutagenesis
(B2YM-Fc and B3YM-Fc). Conversely, the Y–M residues in ephrinB1 were exchanged for the L–W residues (B1LW-Fc); 10 nM, 1 nM, and 0.1 nM of both
wild-type (B1, B2, and B3) and mutant (B1LW, B2YM, and B3YM) ephrin-Fc proteins were tested for their ability to bind NiV-G-HA in an ELISA. The
amount of binding was measured the same as in Figure 1A. The data are averages of three experiments done in triplicates 6 standard error.
DOI: 10.1371/journal.ppat.0020007.g004
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Alternative Receptor for Nipah Virus
ephrinB2- and B3-expressing cells of the subventricular zone
region in an adult mouse [36]. Although the levels of Ephs and
ephrins in other regions of the adult brain are reduced
compared to neonatal-stage expression [27], Eph and ephrins
can alter their expression patterns after injury to the spinal
cord [37], hippocampus [38], or after infection [39,40]. In this
case, NiV infection may alter ephrin expression patterns in
the CNS and disrupt the endogenous Eph–ephrin signaling
resulting in the neuropsychiatric [41] or neuropathologic
sequelae seen in NiV infections.
Zoonotic diseases such as those caused by NiV have become
an increasing threat in several parts of the world [42]. The
habitat of the pteropid fruit bat, considered as the natural
reservoir host, spans from the east coast of Africa across
southern and Southeast Asia, east to the Philippines and
Pacific islands, and south to Australia [43]. Although NiV
outbreaks have only occurred in Malaysia, Bangladesh, and
Singapore, increased surveillance in other geographical
regions of the pteropid habitat found bats to harbor NiV
[44]. Therefore, NiV continues to remain a potential threat to
both human and animal populations. This underscores the
need for the development of antiviral therapeutics. A
complete understanding of Nipah viral entry at the level of
receptor engagement may help in these efforts.
Materials and Methods
Cells and culture conditions. CHO-pgsA745 is a mutant cell line
derived from CHO cells that lack the endogenous expression of
heparin sulfate proteogylcans [19]. CHO-pgsA745 cells and Vero
(African green monkey kidney fibroblasts) cells were maintained in
DMEM/F12 and a-MEM (Invitrogen, Carlsbad, California, United
States), respectively, and both were supplemented with 10% fetal
bovine serum (Omega Scientific, Tarzana, California, United States)
and the antibiotics penicillin and streptomycin. CHO-pgsA745 cells
expressing either wild-type or mutant ephrins were made by selecting
for neomycin resistance with 0.5 mg/ml of G418 after transfection.
Once selected, the ephrin-expressing populations were enriched using
the magnetic bead selection (Miltenyi Biotech, Auburn, California,
United States). Briefly, EphB3-Fc (R & D Systems, Minneapolis,
Minnesota, United States) was coupled to protein G microbeads
(Miltenyi Biotech), and then 2 3 10
6
ephrin-expressing CHO-pgsA745
cells were added. Then, the cell–bead mixture was poured over a
MACS MS column (Miltenyi Biotech), followed by positive cells
elution.
Plasmids and reagents. Soluble Fc-fusion ephrin proteins (eph-
rinA1–A5 and ephrinB1–B3) and Eph proteins (EphA2-Fc and
EphB3-Fc) were purchased from R & D Systems. Human ephrinB2
and ephrinB3 plasmids were purchased from GeneCopoeia (German-
town, Maryland, United States), and human ephrinB1 was obtained
from Open Biosystems (Huntsville, Alabama, Unites States). Each
ephrin open reading frame was subcloned into the pcDNA3.1 vector
(Invitrogen) under CMV promoter-driven expression. In-house
ephrin-Fc fusion constructs were made by subcloning the ectodomain
of each ephrin into the pCR3-Fc vector, which contains the CH2 and
CH3 domains of human IgG1. Mutations in both the full-length
(pcDNA3.1 clones) and soluble (pCR3-Fc clones) [8,45] were made
using the QuikChange (Stratagene, La Jolla, California, United States)
site-directed mutagenesis kit. All subclones and mutations were
confirmed by sequencing.
Binding of soluble ephrins to NiV-G. Supernatant from NiV-G-
HA-transfected 293T cells was used to coat MaxiSorp high protein-
binding 96-well plates (Nalg Nunc International, Rochester, New
York, United States) overnight at 4 8C. The NiV-G-HA-coated plates
were then blocked with 5% bovine serum albumin (BSA) in Tris-
buffer saline (TBS) for 2 h at 37 8C. The plates were rinsed with wash
buffer (1% BSA, 0.05% Tween-20 in TBS), and ephrin-Fc proteins,
diluted in wash buffer, were placed in each well to bind soluble NiV-G
for 1 h at room temperature. The plates were washed three times with
wash buffer and incubated with antihuman Fc monoclonal antibody
conjugated with HRP for 30 min at room temperature. The plates
were then washed three more times, and the amount of bound ephrin
was assessed with 1-step Ultra TMB substrate (Pierce, Rockford,
Illinois, United States). The colorimetric reading was performed on a
spectrophotometer (Dynex Technologies, Chantilly, Virginia, United
States). For each soluble ephrin-Fc, each experiment was performed
three times, each time in triplicates.
Cell surface binding assays. The ephrinB2- and B3-expressing
CHO-pgsA745 cells (CHO-B2 and CHO-B3, respectively) were made
as described above. Increasing amounts of the NiV-G-Fc were
incubated with CHO-B2 or CHO-B3 cells for 1 h on ice. Then, the
cells were washed with buffer and incubated with R-phycoerythrin-
conjugated anti-Fc antibodies for 30 min on ice. The cells were
washed again and fixed with 2% paraformaldehyde, and the data
were collected using a FACScan flow cytometer (Becton Dickinson,
Franklin Lakes, New Jersey, United States). The data were analyzed
using FCS Express V2 (DeNovo Sof tware, Thornhill, Ontario,
Canada). The cell surface K
d
values were calculated using the
GraphPad Prism software (San Diego, California, United States) by
normalizing the highest mean fluorescent intensity value obtained to
100%.
Figure 5. The Leu–Trp Residues in G–H loop of EphrinB3 Are Necessary
for Pseudotyped NiV Entry
(A) The percentage of ephrin cell surface expression (CSE) was measured
by flow cytometry on CHO-pgs745 parental cells (CHO) and CHO-pgs745
cells stably expressing both full-length wild-type ephrins (B1, B2, and B3)
and mutant ephrins (B1LW, B2YM, and B3YM); 10 lg/ml of EphB3-Fc
(solid bar) and 1 nM of NiV-G-Fc (open bar) were used to bind the CHO
cell lines, and the amount of binding was detected the same as in Figure
1B. The data are an average of triplicates 6 standard deviation (SD).
(B) The same CHO cell lines used above were seeded at 10
5
cells per well
and infected with pseudotyped NiV-VSV-DG-Luc virus. The amount of
entry was detected as in Figure 2A. One representative experiment of
three is shown, and data are an average of triplicates 6 SD. In three
independent experiments, the viral entry into B2YM cells was reduced by
45%, 68%, and 85%, respectively, compared to wild-type B2 cells (p ,
0.03, paired t-test).
DOI: 10.1371/journal.ppat.0020007.g005
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Alternative Receptor for Nipah Virus
Pseudotyped virus infection assay. NiV pseudotyped particles were
made from the VSV-DG-Luc virus, a recombinant VSV derived from
a full-length complementary DNA clone of the VSV Indiana serotype
in which the G-protein envelope has been replaced with Renilla Luc
(kindly provided by Andrea Bertolotti-Ciarlet). NiV envelopes F and
G were provided in trans, and the VSV-DG-NiV pseudotyped viruses
were then used to infect the various CHO-pgsA745 cell lines. Briefly,
the cells were seeded overnight in a 48-well plate and then infected
for 1 h at 37 8C with VSV-DG-NiV diluted in 1% FBS in phosphate
buffer saline (PBS). The inoculum was removed and washed with PBS
and replaced by culture media. The next day, the cells were lysed, the
lysates were mixed with Renilla Luc subtrate (Promega, Madison,
Wisconsin, United States), and a luminescence reading was per-
formed on a Veritas luminometer (Turner BioSystems, Sunnyvale,
California, United States). Each condition was done in triplicate and
infections were repeated three times.
Live NiV infection assay. Parental CHO-pgsA745, ephrin-express-
ing CHO-pgsA745, and Vero cells were seeded at 10
5
cells per well in 24-
well plates for live NiV infections. In the L4 facility of the Philipps-
University Marburg under Biosafety Level 4 (BSL-4) conditions, the
cells were then infected with a different MOI of live NiV (kindly
obtained from Andrea Maisner) and incubated for 2 h. The inoculum
was removed, and the cells were washed three times with PBS and left
with 1 ml of DMEM/F12 supplemented with 2% FBS. At 24 h
postinfection, the cells were washed once with PBS and incubated
with 100% ethanol for 10 min at room temperature. After removal of
the ethanol, nuclei were stained with Giemsa solution (1:10 dilution in
water) at room temperature for 30 min. Excess staining solution was
rinsed off with water, and the cells were allowed to dry for 10–15 min
before evaluation of syncytia formation under a light microscope.
Surface plasmon resonance. A BIAcore 3000 instrument (Biacore,
Piscataway, New Jersey, United States) was used to perform binding
kinetics experiments. NiV-G-Fc diluted in sodium acetate (pH 4.0)
was immobilized onto a carboxymethylated dextran (CM5) surface by
standard amine coupling immobilization procedure. The ephrin
analytes were diluted in HBS-EP buffers (BIAcore), and the injections
were performed at a flow rate of 50 ll/min for 180 s. Surfaces were
regenerated using 20 mM sodium hydroxide (BIAcore). Dissociation
constants K
d
,K
on
(association-rate), and K
off
(dissociation-rate) were
determined by fitting binding chromatogram data with BIAcore
evaluation software (version 3.1) using the 1:1 Langmuir binding
model.
Acknowledgments
We thank members of the laboratory for support and encourage-
ment.
Author contributions. OAN and BL conceived and designed the
experiments. OAN, MCW, SE, WW, and SVS performed the experi-
ments and analyzed the data. OAN, HCA, EM, ABC, and RF
contributed reagents/materials/analysis tools. OAN and BL wrote
the paper.
Funding. This work was supported by National Institutes of Health
(NIH) grants (AI059051 and AI060694) to BL; NIH National Research
Service Award grant to OAN (GM07185); a Microbial Pathogenesis
Training grant (AI07323) to MCW; and an emerging infectious
disease grant to ABC. We also acknowledge support to the University
of California Los Angeles flow cytometry core funded through NIH
grants (CA16042 and AI28697).
Competing interests. The authors have declared that no competing
interests exist.
&
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    • "For identifying the most effective mutations, we relied on the G protein structure and previously identified contact residues [22,23,25,26] . Yet, this turned out to be challenging, since the picomolar affinity of G for ephrin-B2 is among the strongest viral envelope-receptor interactions known [40,41]. Accordingly, we found that mutations E533A and W504A, the previously identified key residues for receptor attachment [22,23], were not sufficient to destroy ephrin-B2 binding completely, either individually or in combination (E533A/Q530A and E501A/W504A). "
    [Show abstract] [Hide abstract] ABSTRACT: Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.
    Full-text · Article · Jun 2016
    • "MeV H binds to cell surface molecules CD46, CD150/SLAM or Nectin-4, depending upon virus strain and tissue type (Dorig et al., 1993; Manchester et al., 2000; Muhlebach et al., 2011; Naniche et al., 1993; Noyce et al., 2011; Tatsuo et al., 2000 Tatsuo et al., , 2001). Receptors bound by the NiV and HeV G proteins include Ephrin B2 and Ephrin B3 (Bonaparte et al., 2005; Negrete et al., 2005 Negrete et al., , 2006 ). Viruses classified under the Pneumovirinae subfamily (e.g. "
    [Show abstract] [Hide abstract] ABSTRACT: The Paramyxoviridae include some of the great and ubiquitous disease-causing viruses of humans and animals. In most paramyxoviruses, two viral membrane glycoproteins, fusion protein (F) and receptor binding protein (HN, H or G) mediate a concerted process of recognition of host cell surface molecules followed by fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. The interactions between the F and HN, H or G viral glycoproteins and host molecules are critical in determining host range, virulence and spread of these viruses. Recently, atomic structures, together with biochemical and biophysical studies, have provided major insights into how these two viral glycoproteins successfully interact with host receptors on cellular membranes and initiate the membrane fusion process to gain entry into cells. These studies highlight the conserved core mechanisms of paramyxovirus entry that provide the fundamental basis for rational anti-viral drug design and vaccine development. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015
    • "These results reveal heparan sulfate as a potential therapeutic target for the development of novel approaches against these highly lethal infections. In this context, heparin or its derivatives may be used in a metaphylaxis approach of virus-exposed and potentially infected animals, before the appearance of clinical symptoms, during regular Hendra equine epizootics in Australia (3). [7]) were maintained in F-12 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 0.1 mg streptomycin, 10 mM HEPES, and 2 mM L-glutamine at 37°C in 5% CO 2 . Vero E6, HS-deficient CHO-K1 cells CHO pgsA-745, CHO pgsB-619, and CHO pgsD-677 cells (15), HeLa cells, and stable transfected HeLa cells with phCMV-Nipah- G-Neo and phCMV-Nipah-F-Neo, expressing NiV-G and -F proteins, respectively (HeLa-G and HeLa-F), were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented as described above. "
    [Show abstract] [Hide abstract] ABSTRACT: Nipah virus and Hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus Henipavirus. They infect humans as well as numerous mammalian species. Both viruses use ephrin-B2 and -B3 as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. We have previously reported that Nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. Here, this attachment molecule was identified as heparan sulfate for both Nipah virus and Hendra virus. Cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. Virus pseudotyped with Nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. Furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. Moreover, heparin was shown to bind to ephrin-B3 and to restrain infection of permissive cells in vitro. Consequently, treatment with heparin devoid of anticoagulant activity improved the survival of Nipah virus-infected hamsters. Altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections.
    Full-text · Article · Mar 2015
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