The haparan-sulfate binding motif is important for domain III of
dengue virus binding to BHK21 cells whereas the RGD-like motif is
important for binding to C6/36 cells
Jan-Jong Hung, Meng-Ti Hsieh, Ming-Jer Young and Wen Chang*
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of
Abstract symptoms from mild dengue fever to
Dengue virus belongs to the more severe disease manifestations such
genus Flavivirus that infect mammalin as dengue hemorrhagic fever and dengue
cells through insect vectors such as shock syndrome(15, 23, 25, 67).
mosquito and ticks. Although heparan Dengue virus has a single-stranded,
sulfates were shown to be involved in positive-sense RNA genome that shares
dengue virus absorption to host cells; similar genome organization and virion
however, the structure requirement for structure with other flaviviruses (43).
such interaction remains elusive. This Although Dengue virus infects
study investigated the role of domain III mammalian cells lines of different
of dengue type2 virus envelope protein species in vitro it is transmitted to
in binding to different host cells. humans through mosquito vectors such
Soluble domain III protein that was as Aedes aegypti and Aedes albopictus
purified from bacteria expression system (6).
blocked dengue virus infection to Surface of flavivirus virions is
BHK21 and C6/36 cells. The occupied with viral envelope (E) protein.
interference of domain III in dengue Previous studies indicated that E protein
virus infection is broad in that all four forms dimers on the native virions and
serotypes were inhibited. Furthermore, becomes trimers at low pH and, thus, is
the lab passaged strains as well as the responsible for fusion of virus envelope
clinical virus isolates were blocked by with target cell membrane (1, 28, 75).
the soluble domain III protein. It thus The crystal structure of E protein of tick-
indicated that domain III is involved in borne virus consisting of three domains
dengue virus entry into both mammalian I, II, and III has been determined (64).
and mosquito cells. All flavivirus envelope proteins are
highly homologous so that dengue virus
Introduction E protein could be modeled based on E
Dengue virus is an arthropod- protein of TBEV. Recently, the virion
borne human pathogen that represents a structure of dengue virus was
serious public health threat in tropical determined by using cryoelectron
and subtropical regions (53). The World microscopy and fitting known structure
Health Organization received 500,000 of TBEV-E protein into the electron
dengue fever cases reported by each year density map structure(39). The
and estimates that as many as 50 million conserved arrangements of envelope
people are infected annually(13) and the protein of TBEV, dengue virus and even
reviews within). Dengue virus infections alphavirus indicated that they employ a
give rise to a wide spectrum of disease unique fusion mechanism that is not
shared by other enveloped viruses such from Amersham Pharmacia Biotech AB.
as HIV and HSV(20, 41, 62). A rabbit antiserum against vaccinia virus
In this study, we expressed the virions (1:3,000) was described
domain III portion of dengue type2 virus previously(12). Peptides DV2-1,
(DEN-2) to determine its role in dengue IGVEPGQLKL, are derived from a.a.
virus infection of mammalian BHK21 380-389 of DEN-2 (PL046) sequences.
and mosquito C6/36 cells. Cell binding Peptide DV2-2, EKDSPVN, are derived
assays were performed to mammalian from a.a.360-366 of the same virus.
and mosquito cells in parallel in order to Peptide 2-3 contains a combination of
investigate the structural requirement for DV2-1 and DV2-2 sequences that are
domain III protein interacting with these derived from a.a.360-389. These
two host cells. Our results confirmed peptides were synthesized by ??Inc.
that domain III of E protein is important Chemicals for electrophoresis were
for dengue virus entry into these host purchased from Bio-Rad. Inc.. Other
cells and that different regions of domain chemicals were obtained from Merck
III are required for cell recognition of and Sigma Inc.
mammalian and mosquito cells.
Virus infection interference assays
Material and Methods To test if soluble EIII protein
Viruses and reagents: blocks dengue virus infection at the
Wild-type vaccinia virus (WR) binding step, 5 x 105 BHK-21 cells and
was described before(12), Dengue type 1 C6/36 cells in 60mm dishes were
virus (DEN-1): Hawaii; Dengue type 2 incubated with various amounts of
virus (DEN-2): 16681 and PL046; soluble EIII protein (0, 10 or 100g in
Dengue type 3 virus (DEN-3): H87; 300l) at 4C for 2 h. The cultures were
Dengue type 4 virus (DEN-4): H241 subsequently infected with dengue virus
were obtained from Dr. C.C. King and DEN-1,-2, -3 or -4 at a multiplicity of
were described before (60, 74). infection (MOI ) of 5 PFU per cell at
Japanese Encephalitis Virus (JEV) 4C for 2 h. The infected cultures were
(strain ?) was obtained from Dr. Y.-L. washed and incubated with normal
Lin. BHK-21 cells were maintained in medium at 37C for 24h and the
RPMI medium supplemented with 10% supernatent was harvested for titer
fetal calf serum, 100U of penicillin determination on BHK21 cells.
G/ml, and 100 g of streptomycin/ml.
C6/36, a mosquito cell line established Results
from Aedes albopictus, was obtained Soluble EIII protein of DEN-2 bound
from Dr. C.C. King and maintained in to mammalian BHK21 and mosquito
DMEM/M&M (V/V) medium C6/36 cells.
supplemented with 10% fetal calf serum, The DNA sequences encoding
100U of penicillin G/ml, and 100 g of a.a.295 to a.a.394, i.e. domain III, of E
streptomycin/ml(37). Soluble heparin protein of DEN-2 were amplified by RT-
(HP), chondroitin sulfate (CS), and PCR from purified virion RNA.
dermatan sulfate (DS) were purchased Multiple alignment of amino acid
from Sigma Inc. HiTrapTM Chelating sequences of domain III region with
HP columns and ECLTM Protein several other DEN-2 isolates revealed an
Biotinylation Module were purchased average of 90-95%? identity with
variations limited at few residues at 310, protein and became saturated at 20 g
337, 343, 344 and 366 positions (Figure EIII protein level. The result directly
1A). When other serotypes of dengue demonstrated that EIII protein bound to
viuses were included in alignment the BHK21 cells. The ability of EIII protein
general sequence conservation of dengue binding to mammalian cells is not
virus family? decreased significantly to limited to BHK21 cells since EIII
an average of 43%. Such homology was protein also bound to several other cell
reduced further to 15% along with lines we tested such as SW41 (data not
additional sequence deletion/insertion shown).
events when compared with other When biotinylated EIII protein was
flaviviruses such as TBEV and ?. incubated with mosquito C6/36 cells in
The 300bp DNA fragment was parallel, binding was also observed by
initially cloned into a GST expression FACS (Figure 2B). EIII protein bound to
vector so that the recombinant protein C6/36 cells in a dosage-dependent
was fused with GST at the N-terminus. manner and reached saturation at 25 g
However, the recombinant GST-EIII level. Although cell-associated
fusion protein was in the inclusion fluorescence intensity in C6/36 cells was
bodies and precipitated as insoluble not as strong as in BHK21 cells it was
aggregates (data not shown). clear that EIII protein also is capable of
Alternatively, the DNA fragment was binding to mosquito C6/36 cells.
cloned into a pET expression vector so Fig1:
that the fusion protein, soluble EIII
protein, contain T7 tag sequences at the Domain III of Dengue virus E protein
N-terminus for Ab identification and DEN1
K G M S Y V M C T G S F K L E K E V A E T Q H G T V L V Q V K Y E G T D A P C K I P F S S Q D E K G
hexahistidine sequences at the C-
DEN2 K G M S Y S M C T G K F K V V K E I A E T Q H G T I V I R V Q Y E G D G S P C K I P F E I M D L E K
DEN3 K G M S Y A M C L N T F V L K K E V S E T Q H G T I L I K V E Y K G E H A P C K I P F S T E D G Q G
DEN4 K G M S Y T M C S G K F S I D K E M A E T Q H G T T V V K V K Y E G A G A P C K V P I E I R D V N K
K G M S Y . M C . G F . K E . A E T Q H G T . . . . V . Y E G A P C K I P F D
terminus (Figure 1B). Soluble EIII
DEN1 V T Q N G R L I T A N P I V T D K E K P V N I E A E P P F G E S Y I V V G A G E K A L K L S W F K K
DEN2 R H V L G R L I T V N P I V T E K D S P V N I E A E P P F G D S Y I I I G V E P G Q L K L N W F K K
DEN3 K A H N G R L I T A N P V V T K K E E P V N I E A E P P F G E S N I V I G S G D K A L K I N W Y R K
protein was purified as described in
DEN4 E K V V G R I I S S T P L A E N T N S V T N I E L E P P F G D S Y I V I G V G N S A L T L H W F R K
G R L I T . N P . V T K . P V N I E A E P P F G . S Y I V I G G A L K L . W F . K
materials and methods and was
recognized by an Ab against T7 tag 295 394
Domain III of E protein His-Tag
sequences (Figure 1C, lane 6). The same
protein was also recognized by an pET-21c-EIII
antiserum that recognizes E protein on a
12%SDS-PAGE (Figure 1C, lane 7). Kd
To determine whether soluble EIII 29
protein binds to host cells, EIII protein 18
was biotinylated and tested for its ability
to bind to mammalian BHK-21 and
mosquito C6/36 cells (Figure 2). When
BHK-21 cells were incubated with 1g
of biotinylated EIII protein for 1 h at
4C and analyzed by FACS, binding of
EIII protein to cells was observed, as
evidence by a significant shift in
fluorescence intensity (Figure 2A). The
fluorescence increased further when
cells were treated with 10 g EIII
Fig.2 that soluble HP competitively blocked
EIII protein binding to BHK21 cells.
BHK21 When HP concentration increased to10
or 100g/ml level binding of EIII
protein to BHK-21 cells was diminished
to the background level (Figure 4A). The
inhibitory effect of HP on EIII protein
was specific since neither CS nor DS
affected EIII protein binding to BHK21
cells. The data thus indicated that EIII
protein mainly binds to cell surface HS
on BHK-21 cells.
C6/36 BHK21 cells
Soluble EIII protein bound to heparan
sulfates on BHK-21 cells
Previous studies indicated that
HS on cell surface is important for
dengue virus to attachment to cells and
aa 284- 310 and 386-41 were postulated
to be the HS-binding sites(9). We
therefore tested whether domain III of C6/36 cells
envelope protein mediates dengue virion +
binding to cell surface HS by performing
competition assays with soluble GAGs. EIII
Biotinylated EIII protein was incubated
with BHK21 cells in the presence or in
the absence of soluble GAGs and the +
amount of EIII protein bound to cells
was analyzed by FACS (Figure 4). As EIII
shown in Figure 2A, EIII protein bound
to BHK21 cells and caused a significant
increase of fluorescence intensity with 3
logs of magnitude (Figure 4A). When
1g/ml of soluble HP was incubated
with EIII protein a reduction of
fluorescence was observed, indicating
Soluble E III protein binds to indicated that the RGD-like motif is
mosquito cells through a RGD-like important for EIII protein binding to
motif. C6/36 cells but not to BHK21 cells.
To identify which region other Taken together, Binding of EIII protein
than GAG-binding sites is involved in to BHK21 and C6/36 cells requires
binding of the dengue virus to mosquito different motifs.
cells, we synthesized three peptides that
extend from a.a.360 –366 (DV2-2),
a.a.380-389 (DV2-1) and a.a. 360-389
(DV2-3) of E protein sequences. The DV structure:
reasons of choosing these peptide
regions are several folds. First of all,
these regions are at the solvent
accessible external surface at the lateral
loops of domain III. Second, these
regions contain residues that are
hypervariable and could be subdivided
into distinct groups based on vector
usage, i.e. DV2-2 region. Furthermore,
the DV2-1 region contains a RGD-like Acknowledgements
motif that is conserved in dengue viruses We thank Dr. C.C. King, Kuo and Yi-
and other mosquito-borne viruses and Ling Lin for providing the dengue
has been implicated in cell adhesion (33- viruses.
36). Finally, the DV2-3 peptide is a
longer peptide that covers both DV2-1 References:
and DV2-2 regions and may provide Allison, S. L., J. Schalich, K. Stiasny,
more stability. C. W. Mandl, C. Kunz, and F. X.
We tested whether these peptides Heinz. 1995. Oligomeric rearrangement
blocked EIII protein binding to BHK21 of tick-borne encephalitis virus envelope
and C6/36 cells. Cells were pretreated proteins induced by an acidic pH. J Virol
with these peptides at 100g/ml and 69:695-700.
subsequently incubated with biotinylated
EIII protein. The ability of these Bernfield, M., M. Gotte, P. W. Park,
peptides to block EIII protein binding to O. Reizes, M. L. Fitzgerald, J.
cells was measured by FACS as Lincecum, and M. Zako. 1999.
described in previous sections (Figure7). Functions of cell surface heparan sulfate
In BHK21 cells, EIII protein bound to proteoglycans. Annu Rev Biochem
cells and this fluorescence binding was 68:729-77.
not competed with any of these three
peptides (Figure 7A). In C6/36 cells,
peptide DV2-1but not peptide DV2-2
reduced binding of EIII protein to cells
(Figure 7B). Peptide DV2-3 exhibited no
additional competition and reduced EIII
protein binding to cells to a similar
extent as DV2-1 alone. The data thus