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					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
China.

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


                                            1
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 100g in
Dengue type 3 virus (DEN-3): H87;                300l) at 4C 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)                4C 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 37C 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


                                             2
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
                                                          295
                                                          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
                                                                                                                                                                                                                                                                394
                                                   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
                                                                                                            T7-Tag




protein was also recognized by an                                                                                                       pET-21c-EIII
antiserum that recognizes E protein on a
12%SDS-PAGE (Figure 1C, lane 7).                      Kd
                                                      43
     To determine whether soluble EIII                29
protein binds to host cells, EIII protein             18
was biotinylated and tested for its ability
                                                      14
                                                      6
to bind to mammalian BHK-21 and
mosquito C6/36 cells (Figure 2). When
BHK-21 cells were incubated with 1g
of biotinylated EIII protein for 1 h at
4C 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


                                              3
Fig.2                                           that soluble HP competitively blocked
                                                EIII protein binding to BHK21 cells.
             BHK21                              When HP concentration increased to10
                                                or 100g/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.
                                                Fig.3
               C6/36                            BHK21 cells
                                                                   + EIII




Soluble EIII protein bound to heparan
sulfates on BHK-21 cells
        Previous studies indicated that

                                                         C6/
HS on cell surface is important for
dengue virus to attachment to cells and

                                                         36
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
1g/ml of soluble HP was incubated
with EIII protein a reduction of
fluorescence was observed, indicating



                                            4
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 100g/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



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