(Received for publication, December 23, 1994; and in revised form, May 24, 1995)
From the
Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acid residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.
Dengue virus type 2 (DEN-2), ()a member of the family
of Flaviviridae, has a single-stranded RNA genome of positive-strand
polarity containing 10,723 nucleotides (in New Guinea C strain; (1) ). The 5` end of the genomic RNA has a type I cap, and the
3` end is devoid of a poly(A) tail (for reviews, see (2, 3, 4, 5, 6) ). The
genomic RNA has a single open reading frame containing 10,173
nucleotides encoding a polyprotein of 3391 amino acid residues with a
gene order of 5`-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3`,
which is processed into three structural proteins that are components
of the virion (C, prM, and E) and at least seven nonstructural
proteins, NS1 to NS5, which are expressed in the infected
cells(6) .
The processing of the structural region of the polyprotein is carried out by the host signal peptidase associated with the endoplasmic reticulum(7, 8, 9, 10) . The processing of the nonstructural protein precursors at 2A-2B, 2B-3, 3-4A, and 4B-5 sites containing two basic amino acid residues (such as RR, RK, and KR) followed by a G, A, or S is well characterized, and it requires both NS2B and the N-terminal 180 amino acid residues of NS3 containing the conserved catalytic triad of the serine proteinase family(11, 12, 13, 14, 15, 16, 17, 18, 19, 20) . However, functions of the viral nonstructural proteins in genome replication are not well defined.
The region immediately C-terminal to the catalytic triad of the serine protease domain of NS3 contains conserved segments of NTP-binding proteins and DEAD family of RNA helicases(12, 21, 22) , and in this regard, NS3 appears to be a bifunctional protein. The RNA helicase function of NS3 has not yet been demonstrated, but it has been implicated in an unwinding step of genomic RNA replication, which is thought to occur through the formation of a double-stranded RNA replicative intermediate ( (23, 24, 25) and 42; for a review, see (5) ). NS5, the largest protein of the virus, is the putative RNA-dependent RNA polymerase involved in viral RNA replication based on the conserved motif GDD found in several RNA-dependent RNA polymerases ((26, 27, 28) ; for a review, see (6) and (21) ) and is thought to be involved in the formation of this replicative intermediate form. Thus, NS3 and NS5 are thought to be the components of the putative viral RNA replicase complex. We have overproduced NS3 and NS5 proteins using the recombinant vaccinia virus expression system (29, 30) with a focus on the characterization of these two proteins for their enzymatic activities and for their role in viral RNA replication.
In this report, we demonstrate that NS3 and NS5 proteins interact in vivo in cells infected with either DEN-2 or with recombinant vaccinia viruses encoding these proteins. The NS3 protein also binds in vitro to a C-terminally modified form of NS5 protein immobilized to an affinity matrix. Localization by immunofluorescence using NS3- or NS5-specific antibodies shows that NS3 is localized in the cytoplasm and perinuclear region, whereas NS5 is localized predominantly in the nucleus of DEN-2 infected cells, although diffuse cytoplasmic localization of NS5 is also observed. Immunoprecipitation analysis of the cytoplasmic and nuclear extracts from these cells shows two forms of NS5 in the cytoplasm separable by their electrophoretic mobilities, and only one of these forms is predominantly located in the nucleus. NS3 coimmunoprecipitated with only one form of NS5 in the cytoplasmic extract. Furthermore, we show that NS5 is phosphorylated in vivo at serine residues at multiple sites. The electrophoretic mobilities of the two forms of NS5 are altered upon treatment with a phosphatase, indicating that NS5 exists in differentially phosphorylated states.
Monkey kidney (CV-1), BSC-1 cells, and human TK (143B) cells were grown in Dulbecco's modified
Eagle's medium supplemented with fetal bovine serum (10%). HeLa
cells were grown in suspension culture in Dulbecco's modified
Eagle's medium supplemented with 7% mixed serum (3.5% new born
bovine serum and 3.5% bovine serum). To prepare a monolayer of HeLa
cells, HeLa cells growing in a suspension culture were pelleted, washed
with Dulbecco's modified Eagle's medium containing fetal
bovine serum (10%), and then plated in a suitable flask. DEN-2 virus
(New Guinea strain C) was originally obtained from Walter Reed Army
Institute of Research (Washington, D. C.) and was propagated in CV-1
cells. A laboratory strain of vaccinia virus, WR strain, as well as the
recombinant vaccinia virus encoding the T7 RNA polymerase,
vTF7-3, were originally obtained from Dr. Bernard Moss and then
propagated in HeLa cells(31) .
Figure 1:
NS3 and NS5
interact in vivo. [S]Methionine-labeled
DEN-2-infected cells were lysed and subjected to immunoprecipitation
followed by Western blot analysis (B) and autoradiography (A) of the same blot. Lanes 1 and 6,
immunoprecipitation using preimmune serum; lanes 2 and 4, immunoprecipitation using NS3 antibody (Ab); lanes 3 and 5, immunoprecipitation using NS5
antibody. For Western blot analysis, lanes 1, 2, and 3 were probed with anti-NS5 antibody, and lanes 4, 5, and 6 were probed with anti-NS3 antibody. C, HeLa cells coinfected with recombinant vaccinia viruses
encoding the T7 RNA polymerase gene (vTF7-3) and NS5 (vvNS5) were
lysed, and the cell lysates were immunoprecipitated with anti-NS5
antibody (lanes 2 and 4) and anti-NS3 antibody (lanes 1 and 5). In lanes 3 and 6,
the vTF7-3-infected cell lysates were immunoprecipitated with
anti-NS5 and anti-NS3 antibody, respectively. Immunoprecipitates were
subjected to SDS-PAGE and Western blot analysis. Lanes 1-3 were probed with anti-NS5 antibody, and lanes 4-6 were probed with anti-NS3 antibody. D, the in vitro interaction between NS5.H
and NS3 is shown. Extracts
prepared from HeLa cells coinfected with vTF7-3 and vvNS5.H
were incubated with Ni NTA beads (Qiagen), and the beads were
washed as described under ``Experimental Procedures.'' The
immobilized NS5 containing beads were incubated with an extract
prepared from HeLa cells either infected with the vTF7-3 alone (lane 3) or coinfected with vTF7-3 and vvNS3 (lane
4). Lane 2 was loaded with the extract from
vvNS3-infected HeLa cells without any treatment, and lane 1 was loaded with the sample prepared from the binding of NS3
extract to Ni NTA beads in the absence of NS5.H
. Binding to
Ni NTA beads, washing conditions, SDS-PAGE, and immunoblotting analyses
were carried out as described under ``Experimental
Procedures.''
The results of the experiments described above could
not address the possibility that other DEN-2 proteins might be involved
in facilitating the interaction between NS3 and NS5. In order to
explore this possibility, HeLa cells were coinfected with recombinant
vaccinia viruses encoding NS3 and NS5, and the lysates from these
infected cells were used for immunoprecipitations and immunoblotting
experiments. As the results indicate, NS5 could be coimmunoprecipitated
with NS3 when either anti-NS3 or anti-NS5 antibody was used for
immunoprecipitation (Fig.1C, lanes 1 and 4, respectively). As a negative control, when vTF7-3
(encoding only T7 RNA polymerase)-infected extracts were used for
immunoprecipitation with NS3 or NS5 antibody, no specific bands were
seen (Fig.1C, lanes 3 and 6). These
experiments clearly demonstrated that NS3 and NS5 proteins interact in vivo in DEN-2-infected CV-1 cells or in HeLa cells infected
with recombinant vaccinia viruses encoding NS3 and NS5 proteins. The
results of the latter experiments (Fig.1C) show that
no other viral protein is required for this interaction. In order to
examine whether NS3 and NS5 proteins interact in vitro, we
constructed the recombinant vaccinia virus encoding the NS5 with the
C-terminal modification (vvNS5.H) containing the FLAG
epitope (32) and the histidine tag(33) . The modified
NS5 (NS5.H
) protein expressed in HeLa cells coinfected with
vTF7-3 and vvNS5.H
was immobilized to Ni NTA affinity
beads, which were then washed as described under ``Experimental
Procedures'' to remove any unbound NS5.H
as well as
nonspecific proteins. The NS5 attached to the beads was incubated with
either the extract prepared from the vTF7-3-infected HeLa cells
as negative control or the NS3-containing extracts prepared from HeLa
cells coinfected with vTF7-3 and vvNS3. The beads were washed to
remove any unbound NS3 as well as nonspecific proteins, and the bound
proteins were analyzed by SDS-PAGE and immunoblotting using anti-NS3
antibodies as described under ``Experimental Procedures.'' As
shown in Fig.1D, NS3 was retained in the NS5-bound Ni
NTA beads (lane 4), whereas little or no NS3 was bound to Ni
NTA beads in the absence of immobilized NS5 protein (lane 1)
or when the NS5-bound beads were incubated with the vTF7-3
extract (lane 3). The NS3 extract was loaded into lane 2 as positive control. These experiments indicate that the NS3 and
NS5 proteins interact in vitro.
Figure 2: Subcellular localization of NS3 and NS5 in DEN-2-infected cells. Cells were infected with DEN-2 (50 plaque forming units/cell) at room temperature for 2 h. 36-48 h postinfection, cells were trypsinized and plated on coverslips. Indirect immunofluorescence was performed using anti-NS3 antibody (C) or anti-NS5 antibody (E) as described under ``Experimental Procedures.'' D and F are the respective phase contrast pictures. Mock-infected cells were used as negative controls for anti-NS3 and anti-NS5 antibodies (A and B, respectively).
Figure 3:
NS5 exists in multiple forms. Cytoplasmic (C) and nuclear extracts (N) from
[S]methionine-labeled DEN-2-infected cells were
immunoprecipitated, and the immunoprecipitates were subjected to
SDS-PAGE and autoradiography. Lanes 1 and 2, anti-NS5
antibody; lanes 3 and 4, anti-NS3 antibody; lanes
5 and 6, preimmune serum as negative
controls.
Figure 4:
Multiple forms of NS5 are due to
differential phosphorylation. Cytoplasmic (C) and nuclear (N) extracts from [S]methionine-labeled
DEN-2-infected cells were used for immunoprecipitation with anti-NS5
antibody (Ab). Immunoprecipitated NS5 was either treated with
potato acid phosphatase (lanes 2 and 4) or left
untreated (lanes 3 and 5). Lane 1 is a
negative control where DEN-2-infected whole cell extract was used for
immunoprecipitation with preimmune serum. Immunoprecipitates were
analyzed by SDS-PAGE and autoradiography.
The sensitivity of
immunoprecipitated nuclear and cytoplasmic forms of NS5 to the
phosphatase treatment suggested that the multiple forms of NS5 may be
due to differential phosphorylation. In order to verify this
possibility more directly, CV-1 cells infected with DEN-2 and HeLa
cells infected with recombinant vaccinia virus encoding NS5 were
metabolically labeled using [P]orthophosphate.
Labeled extracts were immunoprecipitated and analyzed by SDS-PAGE
followed by autoradiography (Fig.5). As shown in Fig. 5,
P-labeled NS5 could be immunoprecipitated with anti-NS5
antibody from both the recombinant vaccinia NS5 virus-infected HeLa
cell extracts (lane 2) and the DEN-2-infected CV-1 cell
extracts (lane 3). As a positive control, NS5 was
immunoprecipitated using anti-NS5 antibody from
S-labeled
DEN-2-infected cell extract (Fig.5, lane 1), and as
negative controls, extracts of HeLa cells infected with only the T7 RNA
polymerase gene-encoded recombinant vaccinia virus vTF7-3 were
immunoprecipitated with anti-NS5 antibody (lane 6) or the
extracts used in lanes 2 and 3 with normal rabbit
serum (lanes 5 and 4, respectively). These results
confirm that NS5 is a phosphoprotein. The identity of the
phosphorylated form of NS5 was also verified by immunoblot analysis of
the anti-NS5 immunoprecipitate prepared from
P-labeled
DEN-2-infected cell extracts, followed by autoradiography of the blot
(data not shown).
Figure 5:
Evidence for Phosphorylation of NS5. CV-1
cells infected with DEN-2 or HeLa cells infected with vvNS5 were
metabolically labeled with [P]orthophosphate.
Cell lysates were immunoprecipitated and analyzed by SDS-PAGE and
autoradiography. [
S]Methionine-labeled
DEN-2-infected CV-1 cell lysate (positive control) with anti-NS5
antibody (lane 1); vTF7-3- and vvNS5-coinfected cell
lysate with anti-NS5 antibody (lane 2) or with preimmune serum (lane 5); DEN-2-infected cell lysate with anti-NS5 antibody (lane 3) or with preimmune serum (lane 4);
vTF7-3 infected cell lysate with anti-NS5 antibody (lane
6).
Figure 6:
Phosphoamino acid analysis and
phophopeptide mapping. A, P-labeled NS5 from
recombinant vaccinia NS5-infected cells was acid hydrolyzed and mixed
with unlabeled phosphoserine, phosphothreonine, and phosphotyrosine as
standards. The mixture was subjected to two-dimensional electrophoresis
on thin-layer cellulose-coated plates using buffers at pH 1.9 and 3.5
for the first and second dimensions, respectively. Labeled phosphoamino
acids were detected by autoradiography, and the migration of unlabeled
phosphoamino acids was determined by ninhydrin staining (shown as dotted circles). PS, phosphoserine; PT,
phosphothreonine; PY, phosphotyrosine. Partially hydrolyzed
NS5 is seen as a smear below the phosphoamino acids. B,
P-labeled NS5 was digested with tosylphenylalanyl
chloromethyl ketone-treated trypsin. Tryptic digests were subjected to
electrophoresis in the first dimension and ascending chromatography in
the second dimension on thin-layer cellulose plates. Phosphopeptides
were detected by autoradiography.
The experimental evidence presented in this report indicates
that the two non-structural proteins, NS3 and NS5, interact in vivo both in DEN-2-infected CV-1 cells and HeLa cells infected with
recombinant vaccinia viruses encoding these proteins. Using the
NS5.H immobilized to an affinity matrix (Ni NTA beads), we
show that these two proteins also interact in vitro. This
interaction between NS3 and NS5 supports the notion that they are
components of the putative viral replicase postulated in earlier
studies (6, 42) based on the presence of conserved
motifs of a RNA helicase in NS3 and the G-D-D motif in NS5
characteristic of RNA-dependent RNA
polymerases(12, 26, 28) . The data obtained
from the in vivo and in vitro interaction studies
using the recombinant vaccinia virus-infected cell extracts indicate
that no other flavivirus protein is required for this interaction.
However, a requirement of a cellular protein(s) for this interaction or
participation of other viral and cellular proteins in stabilization of
this complex cannot be ruled out.
Flavivirus genome replication is thought to occur through the formation of double-stranded RNA replicative intermediate(23, 24, 25, 42) . Subcellular localization studies revealed that virus-specific double-stranded RNA appeared to be associated in the perinuclear region of the infected cells(5, 43, 45) . The cell fractionation studies also supported these results and further indicated that viral RNA synthesis appeared to be confined principally to the membranes of the perinuclear endoplasmic reticulum(5, 46, 47, 48, 49, 50) . Both NS3 and NS5 proteins of flavivirus are predicted to have enzymatic roles in flavivirus RNA replication. Although both proteins have been found to be present in the subcellular fractions containing the RNA polymerase activity, direct interaction between these two proteins has not been demonstrated previously.
Previous studies have reported that NS5 of two other flaviviruses is localized in the nucleus and cytoplasm of infected cells(51, 52) . This study, as shown by immunofluorescence, subcellular fractionation, and immunoprecipitation methods, shows that there are two forms of NS5 present in the cytoplasmic fraction that are separable by SDS-PAGE. Only the form with the slower mobility is predominantly located in the nuclear fraction. The multiple forms of NS5 are due to differential phosphorylation, and the hyperphosphorylated form is located predominantly in the nucleus. The presence of two forms of NS5 in DEN-2-infected cells was serentipitously discovered by incubating the infected cells with a medium containing low serum (2.5%). The slower migrating form seems to be the hyperphosphorylated form when compared with the faster migrating and hypophosphorylated cytoplasmic form. Under normal growth conditions containing 10% serum, only one form of NS5 was observed. The result that only the cytoplasmic form of NS5 was found to form a complex with NS3 suggests that differential phosphorylation may regulate the interaction between NS3 and NS5 and thus their participation as components in viral RNA replication.
Based on these results we propose a model as shown in Fig.7.
According to this model, the RNA replicase complex containing NS3 and
NS5 participate in viral RNA replication associated with the
endoplasmic reticulum membrane. In a postreplicative event, NS5 becomes
hyperphosphorylated, dissociates from NS3, and is transported to the
nucleus. It is known that post-translational modification by
phosphorylation regulates protein-protein interactions in eukaryotic
cells (for reviews, see (51) and (52) ). For instance,
in the case of the retinoblastoma gene product (Rb), its state of
phosphorylation is important for its interaction with the cellular
transcription factor E2F (for a review, see (53) and the
references therein). The phosphorylation state of Rb varies in
different stages of the cell cycle. The protein is in a
hypophosphorylated stage at the G/G
phase,
during which it is in a complex with E2F. During the G
/S
phase, Rb gets hyperphosphorylated and dissociates from E2F. Similarly,
phosphorylation of I
B results in the dissociation of
NF
B-I
B complex and translocation of NF
B to the
nucleus(54, 55) .
Figure 7: Model for interaction of NS3 and NS5. NS3 and NS5 interact in the perinuclear region and function as the components of putative viral replicase. Phosphorylation of NS5 by cellular kinase(s) results in the disruption of NS3-NS5 interaction and the transport of NS5 to the nucleus.
The poliovirus RNA-dependent RNA
polymerase, 3D is a phosphoprotein(56) , but the
role of phosphorylation in the function of 3D
is unknown.
For NS5, phosphorylation may play a role in its nuclear transport,
because only the slower migrating hyperphosphorylated form is present
in the nucleus. In this regard, it is worth noting that phosphorylation
has been shown to play a role in the nuclear transport of SV40 large T
antigen(57, 58) . The role of NS5 in the nucleus is
unknown at present. An interesting speculation is that NS5 may regulate
the expression of cellular genes in response to viral infection.